Use of toll-like receptor agonist for treating cancer

ABSTRACT

The present invention is directed to methods and agents used for treating cancer in Toll-Like Receptor 5-expressing tissues by providing a Toll-Like Receptor agonist such as flagellin. The present invention also relates to protecting the liver from a liver toxicity using a Toll-like receptor agonist.

FIELD OF THE INVENTION

This invention relates to methods of treating cancer in Toll-LikeReceptor-expressing tissues, and to methods of protecting the liver fromthe effects of a liver toxicity, using a TLR agonist.

BACKGROUND OF THE INVENTION

Interaction between members of the death receptor family and theircognate ligands induces apoptosis controlling the homeostasis of cellpopulations in tissues, particularly in the immune system. Although manytumor cell types are sensitive to death ligands, activation of Fassignaling also induces massive apoptosis in the liver leading to organfailure and death precluding its use for systemic anticancer therapy.Fas ligand is a 40 kDa physiological agonist of Fas signaling expressedon activated lymphocytes and many tumor cells which can also be secretedthrough metalloproteinase-mediated cleavage and kill the sensitive cellsin autocrine and paracrine manner. Fas is a transmembrane receptorexpressed on activated lymphocytes, variety of tissues and tumor cells.Fas signaling plays crucial role in regulation of the immune system bytriggering autocrine suicide or paracrine death (apoptosis), suppressingimmune reaction by eliminating activated lymphocytes. Upon binding, itinduces p53 independent cell death through extrinsic pathway ofapoptosis engaging DISC formation, caspase-8 and 10, and intrinsic(mitochondrial) apoptosis activating caspase-8 and Bid cleavage, andcytochrome release. Both apoptotic pathways lead to activation ofcaspase-3 and 7. Mitochondrial apoptosis is regulated by pro- andanti-apoptotic Bcl2 family members. In tumor cells, Fas signaling isoften found deregulated either by absence of Fas receptor, or byconstitutive activation of NF-kB resulting in the expression ofanti-apoptotic genes, such as c-Flip, Bcl-2, Bcl-xL. C-Flip, an NF-kBresponsive gene, has been demonstrated to inhibit caspase-8 and Fasmediated apoptosis in tumors (REFs, Kataoka et al 2000).

Upon discovery of p53 independent apoptotic mechanism through Fas, TRAILand TNFα death receptor signaling, they seemed to be promising targetsfor anti-cancer therapy since tumor cells usually have impaired p53function. A severe hepatotoxicity, however, is induced by death receptorligands. This has hampered development of these anti-cancer therapies.While Fas agonists cause liver damage and TNF-a induces stronginflammation in liver, lungs and other organs, TRAIL is the least toxicin humans. TRAIL has therefore received more attention than otheragonists for the clinical application for an anticancer treatment. Manytumors, however, are not sensitive to TRAIL therapy. Several approachesto resolve death receptor toxicity issue are currently undertaken, mostof which are aimed to increase tumor sensitivity by blockage of NF-kBactivity and increasing receptor expression thus reducing the amount ofdrug necessary for the effective therapy. Another direction is tolocalize the drug delivery to the tumors to minimize toxic effects ondistant organs. To date, there is no reliable approach to the preventionof toxicity (including liver injury) that would allow the systemicapplication of death receptor agonists in clinical trials. Accordingly,there is a need in the art for methods of preventing the undesirableeffects of death receptors when they are used to treat cancer. Inparticular, there is a need to protect the liver from these undesirableeffects. There is also a need for protecting the liver from livertoxicities in general.

TLRs are found to be expressed on both epithelial and endothelial cellsas well as immunocytes. At present, thirteen TLRs have been identifiedin mammals. Upon receptor stimulation, several common signaling pathwaysget activated such as NF-kB, AP-1, PI3K/AKT and mitogen-activatedprotein kinases (MAPK) leading to increased survival, stimulation ofcell proliferation and the secretion of many cytokines with chemotacticand pro-inflammatory functions. Induction of TLR in cancer cells can beused to treat cancer, however, the distribution of different TLRs variessignificantly among the various organs and cell types. This affects thecytokine profile and extent of the inflammatory response of cells.Accordingly, there is a need in the art for cancer immunotherapeuticmethods that do not depend on the presence of TLR5 expression.

SUMMARY OF THE INVENTION

Provided herein is a method of treating cancer in a mammal, which maycomprise administering to a mammal in need thereof of Toll-Like Receptor(TLR) agonist. Also provided is a method of reducing cancer recurrencein a mammal, which may comprise administering to a mammal in needthereof a TLR agonist. The cancer may be present in a tissue thatexpresses TLR. The cancer may be a metastasis or tumor regrowth.

The TLR agonist may be flagellin. The cancer may not express TLR, whichmay be TLR5. The tissue may be liver, lung, bladder, or intestinal. Thecancer may be metastatic. The cancer may be melanoma, colon, breast,prostate, or a hematological malignancy, which may be lymphoma. Thecancer may be tumor.

The agent may be administered as a monotherapy. The mammal may not bereceiving a combination therapy. The mammal may also not be receivingchemotherapy or radiation therapy, but may be treated surgically. Themammal may have sufficient innate immunity, which may be at a level thatis equivalent to the level required for eligibility for a first orsubsequent round of chemotherapy. The mammal may have a white blood cellcount within the range of normal, or may have a white blood cell countindicative of mild-immunosuppression. The TLR agonist may beadministered to the mammal before, after or concurrent with removal of atumor. The TLR agonist may be administered during tumor removal.

Further provided herein is a method of treating cancer in a mammal,which may comprise administering to a mammal in need thereof a FASagonist and a TLR agonist, which may be flagellin. The FAS agonist maybe a FAS agonist antibody. The cancer may be metastatic, and may be atumor. The cancer may not express a TLR. The cancer may havemetastasized to an invaded tissue that expresses TLR. The invaded tissuemay be liver, bladder, lung, or intestinal.

Also provided herein is a method of protecting liver tissue in a mammalfrom the effects of a liver toxicity, which may comprise administeringto a mammal in need thereof a TLR agonist. The toxicity may be a FASligand, a FAS agonistic antibody, TNFα, acetaminophen, alcohol, a viralinfection of the liver, or a chemotherapeutic agent. The toxicity mayalso be a Salmonella infection, which may be from Salmonellatyphimurium. The TLR agonist may be flagellin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the domain structure of bacterial flagellin. The Cabackbone trace, hydrophobic core distribution and structural informationof F41. Four distinct hydrophobic cores that define domains D1, D2a, D2band D3. All the hydrophobic side-chain atoms are displayed with the Cabackbone. Side-chain atoms are color coded: Ala, yellow; Leu, Ile orVal, orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms).c, Position and region of various structural features in the amino-acidsequence of flagellin. Shown are, from top to bottom: the F41 fragmentin blue; three b-folium folds in brown; the secondary structuredistribution with a-helix in yellow, b-structure in green, and b-turn inpurple; tic mark at every 50th residue in blue; domains D0, D1, D2 andD3; the axial subunit contact region within the proto-element in cyan;the well-conserved amino-acid sequence in red and variable region inviolet; point mutations in F41 that produce the elements of differentsupercoils. Letters at the bottom indicate the morphology of mutantelements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V),R-type straight; C (D313Y, A414V, A427V, N433D), curly33.

FIG. 2 shows a schematic of Salmonella flagellin domains, its fragments,and its interaction with TLR5. Dark bars denote regions of the flagellingene used to construct fragments comprising A, B, C, A′ and B′.

FIG. 3 depicts flagellin derivatives. The domain structure andapproximate boundaries (amino acid coordinates) of selected flagellinderivatives (listed on the right). FliC flagellin of Salmonella dublinis encoded within 505 amino acids (aa).

FIG. 4 shows the nucleotide and amino acid sequence for the followingflagellin variants: AA′ (SEQ ID NO: 7-8), AB′ (SEQ ID NO: 9-10), BA′(SEQ ID NO: 11-12), BB′ (SEQ ID NO: 13-14), CA′ (SEQ ID NO: 15-16), CB′(SEQ ID NO: 17-18), A (SEQ ID NO: 19-20), B (SEQ ID NO: 21-22), C (SEQID NO: 23-24), GST-A′ (SEQ ID NO: 25-26), GST-B′ (SEQ ID NO: 27-28),AA′n1-170 (SEQ ID NO: 29-30), AA′n1-163 (SEQ ID NO: 33-34), AA′n54-170(SEQ ID NO: 31-32), AA′n54-163 (SEQ ID NO: 335-36), AB′n1-170 (SEQ IDNO: 37-38), AB′n1-163 (SEQ ID NO: 39-40), AA′n1-129 (SEQ ID NO: 41-42),AA′n54-129 (SEQ ID NO: 43-44), AB′n1-129 (SEQ ID NO: 45-46), AB′n54-129(SEQ ID NO: 47-48), AA′n1-100 (SEQ ID NO: 49-50), AB′n1-100 (SEQ ID NO:51-52), AA′n1-70 (SEQ ID NO: 53-54) and AB′n1-70 (SEQ ID NO: 55-56). ThepRSETb leader sequence is shown in Italic (leader includes Met, which isalso amino acid 1 of FliC). The N terminal constant domain isunderlined. The amino acid linker sequence is in Bold. The C terminalconstant domain is underlined. GST, if present, is highlighted.

FIG. 5 shows a comparison of amino acid sequences of the conserved amino(FIG. 5A) and carboxy (FIG. 5B) terminus from 21 species of bacteria.The 13 conserved amino acids important for TLR5 activity are shown withshading. The amino acid sequences are identified by their accessionnumbers from TrEMBL (first letter=Q) or Swiss-Prot (first letter=P).

FIG. 6 shows the sequence of human TLR5.

FIG. 7 NF-kB activation in vivo in response to CBLB502 and LPSinjections. A. Background and NF-kB dependent luciferase expression inBALB/c-Tg (IκBα-luc)Xen reporter mice was detected by noninvasiveimaging 2 hs after the treatment with CBLB502 (0.2 mg/kg). B. NF-kBdependent luciferase expression in liver, small intestine (ileum part),colon, spleen, kidneys, lungs and heart was assessed in the reportermice 2 hs after s.c. injections of 100 μl of either PBS, CBLB502 (0.2mg/kg) or LPS (1 mg/kg). Luciferase activity normalized per μg of theprotein extract was detected in 3 mice in each group. Bars representaverage+/−s.d. C. The dynamics of NF-kB nuclear translocation (p65)indicative of the bioactivity of agonists LPS and CBLB502 in liver fromNIH-Swiss mice injected s.c either with CBLB502 or LPS. Control micewere injected with PBS. Tissue samples were obtained 20, 40 and 60 minafter the treatments, processed into paraffin blocks. Nucleartranslocation of p65 in primary mouse hepatocytes isolated fromNIH-Swiss mice (D) and human hepatocytes purchased from (BD Biosciences)(E) was detected after in vitro treatment with CBLB502 (100 ng/ml) orLPS (1 μg/ml) for indicated period of time. Control hepatocytes remainedintact. P65 was stained with green fluorescence, cytokeratin-8 with redfluorescence and nuclei with non-specific Dapi blue staining. Picturesare taken at ×20 magnification. Arrows indicate Kupffer and endothelialcells determined based on morphological criteria.

FIG. 8 shows CBLB502 protection from Fas mediated hepatotoxicity. A.Survival of NIH-Swiss mice after i.p. injection of 4 μg of anti-Fasantibodies alone or in combination with CBLB502 (1 μg/mouse) injected 30min, 2 hours and 6 hours prior antibodies. In parenthesis are thenumbers of mice per each treatment. B. Protection of livers fromanti-Fas antibody toxicity. Apoptosis in livers 5 hours after injectionsof anti-Fas antibodies was detected using TUNEL technique. C. Tissuemorphology with H&E staining revealed necrotic damage to livers byanti-Fas antibody injections and protection by CBLB502. D. Hemorrhage inliver was detected using erythrocyte autofluorescence (rhodaminechannel, red), mouse IgG control (Cy5-conjugated anti-mouse IgGantibody, pceudocolored in purple) and DAPI nuclei (blue). E.Caspase-3/7 activity in liver samples of NIH-Swiss mice was determinedin tissue protein lysates 5 hours after injection of 3 μg anti-Fasantibody with or without CBLB502 thirty minute pre-treatment. N=3. Barsrepresent average+/−s.d. F. Alanine aminotransferase (ALT) accumulationin blood serum of NIH-Swiss mice was detected 5 hours after anti-Fasantibody injections with or without CBLB502. N=3. Bars representaverage+/−s.d. G. Caspase-8 activity in liver samples of NIH-Swiss micewas determined in tissue protein lysates 5 hours after injection of 3 μganti-Fas antibody with or without CBLB502 thirty minute pre-treatment.N=3. Bars represent average+/−s.d.

FIG. 9 shows regulation of apoptosis-related factors by CBLB502 in liverand its effect on Fas-mediated antitumor activity in CT-26 tumor model.Inhibition of caspase-8 (A) and Bid (B) cleavage by CBLB502 detected inliver isolated from C57BL/6 mice 2 hours after anti-Fas antibodyinjections (5 μg) alone or in combination with CBLB502 by western blot.C. RNA expression of Bcl2A1B, Bcl2A1D, IER-3, Fos, Jun and JunB genes inlivers of intact mice and treated with CBLB502 for 30 min and 2 hourswas detected by RT-PCR. GAPDH was used as a control to monitor theinduction of gene expression. D. Mice with s.c. growing CT-26 tumorswere injected either with single anti-Fas antibodies (4 μg/mouse) andCLB502 or their combination. Control mice (“intact”) received PBS inreplace of CBLB502 and antibodies. In parenthesis are the numbers oftumors in each group. The results represent the average tumor volumes(m+/−standard error). (*)—The difference between intact and combinationtreatment groups is significant (p<0.05). E. Mice were treated withanti-Fas antibodies alone or in combination with CBLB502 on day 5 afterintrasplenic injection of luciferase expressing CT-26 tumor cells. Tumorgrowth in livers was determined using Xenogen IVIS Imaging System on thedays 10, 15, 17, 22, 28 and 40 after tumor cell inoculation. Images of 3mice from each group taken on day 15 are presented. The differencebetween proportions of mice with tumor-free livers in CBLB502-treatedand control groups reaches statistical significance (p<0.05) on daysindicated by asterisks. F. Migration and infiltration of immunocytes(arrows) into tumor nodules grown in liver of mice 5 hrs post treatmentwith CBLB502.

FIG. 10. Dynamics of NF-kB activation in different organs afterinjections with CBLB502 (5 μg, s.c.) or LPS (20 μg, s.c.). Mice wereeuthanized 2, 6, 24 and 48 hours later by CO2 inhalation. Luciferaseactivity in protein extracts from liver, large intestine, kidneys andlungs was normalized per μg of the protein extract and average valueswere calculated per organ. Luciferase fold induction was calculated asratio between average luciferase activity in protein extract from organsof the TLR agonist treated mice and that obtained in the extracts fromthe corresponding organs of the PBS injected control mice (3mice/group). Bars represent fold induction as average±s.e.

FIG. 11. NF-kB dependent luciferase expression in primary culture ofmouse hepatocytes isolated from luciferase reporter mice and treated invitro for 3 hours with CBLB502 (100 ng/ml), LPS (5 μg/ml) or PBScontrol. Then hepatocytes were rinsed with PBS and collected in celllysis buffer (Promega). Luciferase activity in the protein supernatantswas determined by Promega reporter system and normalized per μg of theprotein extract. Bars represent luciferase units (mean±s.d.).

FIG. 12. H&E staining of liver samples from NIH-Swiss mice treated withCBLB502, anti-Fas antibodies (3 μg) or their combination obtained atdifferent time-points after the treatment. Samples of livers wereobtained 5, 12 and 26 hours after injections of anti-Fas antibodies,fixed in 10% formalin, embedded in paraffin and stained for tissuemorphology with hematoxilin and eosin.

FIG. 13. TLR5 expression on B16 and CT-26 cells. A. Expression of mRNAencoding TLR5 and GAPDH (as a control) genes in B16 and CT-26 cells weredetermined by RT-PCR. A region of mouse TLR5 mRNA was amplified usingprimers specific for the mouse TLR5 gene: forward(5′-AGTCCCCCAGCTCCAGTTTC-3′) and reverse (5′-GGAGCCCCCTAGCAGTG AGT-3′).B. NF-kB activation in B16 and CT-26 tumor cells in response to CBLB502,mouse TNFα and LPS was tested using luciferase reporter assay. Datarepresent luciferase units (mean±s.d).

FIG. 14. Shows TLR5 expresion in CT-26 tumor cells and A20 lymphomacells. For FIGS. 14A and C, total RNA was extracted from CT-26 and B16tumor cells (FIG. 14A) and CT-26 and A20 cells (FIG. 14C) using TRIzolreagent as described in the main text of the manuscript. The primers forTLR5 were designed using LaserGene software (DNASTAR, Inc., Madison,Wis.). A region of mouse TLR5 mRNA (GenBank Accession No.NM_(—)016928.2) was amplified using primers specific for the mouse TLR5gene: forward (5′-AGTCCCCCAGCTCCAGTTTC-3′) and reverse(5′-GGAGCCCCCTAGCAGTGAGT-3′). GAPDH was used as a control to monitor theinduction of gene expression. cDNAs were synthesized using Superscript™II Reverse Transcriptase and oligo(dT)12-18 primer (Invitrogen,Carlsbad, Calif.). B. An in vitro luciferase assay for NF-kB activationin B16 (TLR5 positive) and CT-26 TLR5 negative) tumor cells wasperformed.

FIG. 15 shows the dynamics of TLR5 positive HCT116 tumor growth inathymic nude mice after CBLB502 or PBS (no treatment) treatments (0.2mg/kg, s.c., days 1, 2, 3), n=6-10.

FIG. 16 shows 293-TLR5 tumor growth in athymic nude mice after CBLB502or PBS (no treatment) treatments (0.2 mg/kg, s.c., days 1, 2, 3),n=6-10.

FIG. 17 shows the dynamics of xenogenic A549 tumor growth in athymicnude mice during 2 courses of CBLB502 vs. PBS (control) treatments (days1, 2, 3, 14, 15 and 16), n=6-10. Antitumor activity of colon HCT116adenocarcinoma s.c. Grown as a xenograft in athymic mice. HCT116 wereinjected s.c. into 2 flanks of 8 athymic nude mice (0.5×106/100 ml ofPBS) to induce tumors. When tumors became of about 3-5 mm in diameter(by day 6 after injections) mice were randomly distributed into 2groups, 5 mice for CBLB502 treated group and 3 mice in PBS controlgroup.

FIG. 18 shows the rate of SCCVII orthotopic tumor growth in syngenic C3Hmice after CBLB502 or PBS (no treatment) treatments (0.1 mg/kg, s.c.days 1, 2, 3) to reach 400 mm³ tumor size, n=6-10. Right figurerepresents the amount of days needed for tumors to reach 400 mm3 volumewith and without treatment with CBLB502.

FIG. 19. Fischer rats with s.c. growing syngeneic Ward colon tumors weretreated with CBLB502 (0.2 mg/kg) was administered by i.p. once a day forthree days.

FIG. 20 shows the dynamics of xenogenic A549-shV and A549-shTLR5 tumorgrowth in athymic nude mice after CBLB502 or PBS (control) treatments(days 1, 2, 3). Statistical difference between tumor volumes on days 2,4, 6 and 8 observed in A549-shV tumors (p<0.05), n=9-14. Right figuredemonstrates NF-kB dependent induction of luciferase reporter expressionin A549-shV and A549-shTLR5 in response to CBLB502 treatment.

FIG. 21 shows the dynamics of H1299 (control) and H1299-TLR5 tumorgrowth in athymic nude mice after CBLB502 or PBS (control) treatments(days 1, 2, 3), n=6. Right figure demonstrates IL-8 production inresponse to CBLB502 treatment as indicative of TLR5 function inH1299-TLR5 cells.

FIG. 22 shows that the bladder strongly responds to CBLB502.

FIG. 23 shows that CBLB502 treatment delays tumor appearance and growthin livers, even in tumors that do not express TLR5.

FIG. 24 shows CBLB502 protection from Fas mediated hepatotoxicity.

FIG. 25 shows that the liver is protected from TNFα and LPS toxicity byCBLB502.

FIG. 26 shows that CBLB502 protects the lungs from TNF and LPS toxicity.

FIG. 27 shows that CBLB502 protects mice from legal oral administrationof Salmonella.

FIG. 28 shows that irinotecan abrogates the antitumor effect offlagellin (CBLB502).

DETAILED DESCRIPTION

The inventors have made the surprising discovery that the provision of aToll-Like Receptor (TLR) agonist, such an agonist of TLR5 likeflagellin, can effectively inhibit the growth of and reduce cancercells, even when the cells do not express TLR5. The TLR agonist may beparticularly useful in treating liver, bladder, lung, and intestinalcancers, whether primary or metastatic, as well as cancer affectingother TLR5-positive tissues. The TLR agonist can also be used to treatcancers that originate in tissues other than the liver, bladder, lung,intestinal, and other TLR5-positive tissues, but metastasize to thesetissues. Even though the metastatic cancer cells do not express TLR5,the cancer may nonetheless be treatable with the TLR agonist when thecancer has metastasized to TLR5-expressing tissues such as the liver.While not being bound by theory, the idea implemented in this inventionis that TLR agonists effectively reduce or kill cancer cells affecting atissue that has a strong innate immunity system, thereby obviating theneed for any pre-existing expression of TLR5 in the cancer cells.Unexpectedly, by providing a TLR agonist, the innate immune system issufficiently triggered so as to treat cancers that are devoid of TLR5expression. Thus, TLR5 does not need to be provided to the cancer cellsin order for the TLR agonist to effectively reduce or kill cancer cells.

The inventors have also made the surprising discovery that a TLR agonistcan protect the liver from a liver toxicity. For example, death ligandsand activators of FAS-mediated apoptosis, such as FAS ligand andanti-FAS agonistic antibodies, can induce does-dependent hepatotoxicity.Administering the TLR agonist can protect the liver against suchtoxicities. This unexpected property of TLR agonists allows it becombined with FAS agonists or TNF for cancer treatment, such that theadverse of effects of the FAS agonist or TNF are reduced or prevented.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

“Administer” may mean a single dose or multiple doses of an agent oragent.

“Analog” may mean, in the context of a peptide or polypeptide, a peptideor polypeptide comprising one or more non-standard amino acids or otherstructural variations from the conventional set of amino acids.

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, or derivatives thereof, including Fab, F(ab′)2, Fd, andsingle chain antibodies, diabodies, bispecific antibodies, bifunctionalantibodies and derivatives thereof. The antibody may be a monoclonalantibody, polyclonal antibody, affinity purified antibody, or mixturesthereof which exhibits sufficient binding specificity to a desiredepitope or a sequence derived therefrom. The antibody may also be achimeric antibody. The antibody may be derivatized by the attachment ofone or more chemical, peptide, or polypeptide moieties known in the art.The antibody may be conjugated with a chemical moiety.

A “derivative” may mean a peptide or polypeptide different other than inprimary structure (amino acids and amino acid analogs). Derivatives maydiffer by being glycosylated, one form of post-translationalmodification. For example, peptides or polypeptides may exhibitglycosylation patterns due to expression in heterologous systems. If atleast one biological activity is retained, then these peptides orpolypeptides are derivatives according to the invention. Otherderivatives may include fusion peptides or fusion polypeptides having acovalently modified N- or C-terminus, PEGylated peptides orpolypeptides, peptides or polypeptides associated with lipid moieties,alkylated peptides or polypeptides, peptides or polypeptides linked viaan amino acid side-chain functional group to other peptides,polypeptides or chemicals, and additional modifications as would beunderstood in the art.

A “fragment” may mean a portion of a reference peptide or polypeptide.

A “homolog” may mean a peptide or polypeptide sharing a commonevolutionary ancestor.

A “leader sequence” may be a nucleic acid encoding any peptide sequencethat is linked and translated with a peptide or polypeptide of interestto allow the peptide or polypeptide of interest be properly routedthrough a eukaryotic cell's endoplasmic reticulum and Golgi complexesfor the purposed of extracellular secretion from the cell's membrane.The leader peptide sequence may be derived from alkaline phosphatase.The leader sequence may have a DNA sequence comprisingatgctgctgctgctgctgctgctgggcctgaggctacagctct ccctgggc.

A “liposome” may mean a tiny bubble (vesicle) made out of the samematerial as a cell membrane. A liposome be filled with drugs and used todeliver drugs for cancer and other diseases. A liposome may be filledwith a vector. A liposome membrane may be made of phospholipids, whichare molecules that have a head group and a tail group. The head of theliposome may be attracted to water, and the tail, which is made of along hydrocarbon chain, is repelled by water. The tails may be repelledby water, and line up to form a surface away from the water. The lipidsin the plasma membrane may be chiefly phospholipids likephosphatidylethanolamine and phosphatidylcholine. Liposomes may becomposed of naturally-derived phospholipids with mixed lipid chains(like egg phosphatidylethanolamine), or of pure surfactant componentslike DOPE (dioleoylphosphatidylethanolamine).

A “peptide” or “polypeptide” may mean a linked sequence of amino acidsand may be natural, synthetic, or a modification or combination ofnatural and synthetic.

“Substantially identical” may mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100 amino acids.

“Treating,” “treatment,” or “to treat” each may mean to alleviate,suppress, repress, eliminate, prevent or slow the appearance ofsymptoms, clinical signs, or underlying pathology of a condition ordisorder on a temporary or permanent basis. Preventing a condition ordisorder involves administering a agent of the present invention to asubject prior to onset of the disease. Suppressing a condition ordisorder involves administering a agent of the present invention to asubject after induction of the condition or disorder but before itsclinical appearance. Repressing the condition or disorder involvesadministering a agent of the present invention to a subject afterclinical appearance of the disease.

A “variant” may mean means a peptide or polypeptide that differs inamino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include theability to bind to a toll-like receptor and to be bound by a specificantibody. Variant may also mean a protein with an amino acid sequencethat is substantially identical to a referenced protein with an aminoacid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporatedfully herein by reference. Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art.Substitutions may be performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hyrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.

A “vector” may mean a nucleic acid sequence containing an origin ofreplication. A vector may be a plasmid, a yeast or a mammalianartificial chromosome. A vector may be a RNA or DNA vector. A vector maybe either a self-replicating extrachromosomal vector or a vector whichintegrates into a host genome.

2. TOLL-LIKE RECEPTOR AGONIST

Provided herein is a TLR agonist. The TLR agonist may be a PAMP, whichmay be conserved molecular product derived from a pathogen. The pathogenmay be a Gram-positive bacterium, Gram-negative bacterium, fungus, orvirus. The TLR agonist may be a damage-associated molecular pattern(DAMP) ligand, which may be an endogenous molecule released from injuredor dying cells. A DAMP or PAMP may initiate an immune response throughTLR signals and recruit adapter molecules within the cytoplasm of cellsin order to propagate a signal. The TLR agonist may be an agonist forthe TLR, which may be a ligand from the following in Table 1:

TABLE 1 TLRs and Ligands TLR Ligand DAMP Ligand PAMP TLR1 Triacyllipoproteins TLR2 Heat Shock proteins Peptidoglycan HMGB1 (high mobilityLipoprotein group box 1-amphoterin) Lipoteichoic acid Zymosan TLR3 SelfdsRNA Viral dsRNA TLR4 Heat shock proteins Heat shock proteinsFibrinogen Lipopolysaccharides Heparan sulfate RSV fusion proteinFibronectin MMTV (Mouse mammary tumor virus) envelope proteinsHyaluronic acid Paclitaxel HMGB1 TLR5 flagellin TLR6 Lipoteichoic acidTriacyl lipoproteins zymosan TLR7/TLR8 Self ssRNA Viral ssRNA TLR9 SelfDNA Bacterial and viral DNA TLR10 TLR11 Profilin

The TLR agonist may be a fragment, variant, analog, homology orderivative of a PAMP or DAMP that binds a TLR and induces TLR-mediatedactivity, such as activation of NF-κB activity. The TLR agonsistfragment, variant, analog, homolog, or derivative may be at least 30-99%identical to amino acids of a TLR-agonist and induce TLR-mediatedactivity.

The TLR agonist may target a TLR such as TLR-5. The TLR agonist may bean agonist of TLR-5 and stimulate TLR-5 activity. The TLR agonist may bean anti-TLR5 antibody or other small molecule. The TLR agonist may beflagellin.

The flagellin may also be a flagellin or flagellin-related polypeptide.The flagellin may be from any source, including a variety ofGram-positive and Gram-negative bacterial species. The flagellin may bea flagellin polypeptide from any Gram-positive or Gram-negativebacterial species including, but not limited to, a flagellin polypeptidedisclosed in U.S. Pat. Pub. No. 2003/000044429, the contents of whichare fully incorporated herein by reference. For example, the flagellinmay have an amino acid sequence from a bacterial species depicted inFIG. 7 of U.S. Patent Publication No. 2003/0044429. The nucleotidesequences encoding the flagellin polypeptides listed in FIG. 7 of U.S.2003/0044429 are publicly available at sources including the NCBIGenbank database. The flagellin may also be a flagellin peptidecorresponding to an Accession number listed in the BLAST results shownin FIG. 25 of U.S. Patent Pub. 2003/000044429, or a variant thereof. Theflagellin may also be a flagellin polypeptide as disclosed in U.S.Patent Appl. Publication No. 2009/0011982, the contents of which arefully incorporated herein. The flagellin maybe any one of a flagellinpolypeptide as disclosed in FIGS. 3 and 4 herein.

The flagellin may be a fragment, variant, analog, homology or derivativeof a flagellin that binds TLR5 and induces TLR5-mediated activity, suchas activation of NF-κB activity. A fragment, variant, analog, homolog,or derivative of flagellin may be at least 30-99% identical to aminoacids of a flagellin that binds TLR5 and induces TLR5-mediated activity.

The flagellin may be from a species of Salmonella, a representativeexample of which is S. dublin (encoded by GenBank Accession NumberM84972). The flagellin related-polypeptide may be a fragment, variant,analog, homolog, or derivative of M84972, or combination thereof, thatbinds to TLR5 and induces TLR5-mediated activity, such as activation ofNF-kB activity. A fragment, variant, analog, homolog, or derivative offlagellin may be obtained by rational-based design based on the domainstructure of Flagellin and the conserved structure recognized by TLR5.

The flagellin may comprise at least 10, 11, 12, or 13 of the 13conserved amino acids shown in FIG. 2 (positions 89, 90, 91, 95, 98,101, 115, 422, 423, 426, 431, 436 and 452). The flagellin may be atleast 30-99% identical to amino acids 1 174 and 418 505 of M84972. FIG.26 of U.S. Patent Appl Publication No. 2009/0011982, the contents ofwhich are fully incorporated herein, lists the percentage identity ofthe amino- and carboxy-terminus of flagellin with known TLR-5stimulating activity, as compared to M84972.

The flagellin may be the major component of bacterial flagellum. Theflagellin may be composed of three domains (FIG. 1). Domain 1 (D1) anddomain 2 (D2) may be discontinuous and may be formed when residues inthe amino terminus and carboxy terminus are juxtaposed by the formationof a hairpin structure. The amino and carboxy terminus comprising the D1and D2 domains may be most conserved, whereas the middle hypervariabledomain (D3) may be highly variable. Studies with a recombinant proteincontaining the amino D1 and D2 and carboxyl D1 and D2 separated by anEscherichia coli hinge (ND1-2/ECH/CD2) indicate that D1 and D2 may bebioactive when coupled to an ECH element. This chimera, but not thehinge alone, may induce IkBa degradation, NF-kB activation, and NO andIL-8 production in two intestinal epithelial cell lines. Thenon-conserved D3 domain may be on the surface of the flagellar filamentand may contain the major antigenic epitopes. The potent proinflammatoryactivity of flagellin may reside in the highly conserved N and C D1 andD2 regions (See FIG. 1).

The flagellin may induce NF-kB activity by binding to Toll-like receptor5 (TLR5). The TLR may recognize a conserved structure that is particularto the flagellin. The conserved structure may be composed of a largegroup of residues that are somewhat permissive to variation in aminoacid content. Smith et al., Nat Immunol. 4:1247-53 (2003), the contentsof which are incorporated herein by reference, have identified 13conserved amino acids in flagellin that are part of the conservedstructure recognized by TLR5. The 13 conserved amino acids of flagellinthat may be important for TLR5 activity are shown in FIG. 2.

Numerous deletional mutants of flagellin have been made that retain atleast some TLR5 stimulating activity. The flagellin may be such adeletional mutant, and may be a deletional mutant disclosed in theExamples herein. The flagellin may comprise a sequence translated fromGenBank Accession number D13689 missing amino acids 185-306 or 444-492,or from GenBank Accession number M84973 missing amino acids 179-415, ora variant thereof.

The flagellin may comprise transposon insertions and changes to thevariable D3 domain. The D3 domain may be substituted in part, or inwhole, with a hinge or linker polypeptide that allows the D1 and D2domains to properly fold such that the variant stimulates TLR5 activity.The variant hinge elements may be found in the E. coli MukB protein andmay have a sequence as set forth in International Application No.PCT/US10/51646, filed on Oct. 6, 2010, the contents of which areincorporated herein by reference.

The flagellin as described above may further comprise a leader sequence.The flagellin further comprising a leader sequence may be CBLB502S.

3. AGENT

This invention also relates to an agent comprising a therapeuticallyeffective amount of a TLR agonist. The agent may be a polypeptide. Theagent may also be a vector. The vector may comprise a nucleic acidencoding the TLR agonist. The vector may be capable of transducingmammalian cells. The vector may be delivered into a mammalian cell by avirus or liposome related vector system. The virus vector system may bean adenovirus or a cytomegalovirus.

The agent may be a liposome harboring the vector. The liposome maybecapable of transducing mammalian cells and delivering the vector forexpression.

The agent may be a drug formulation that activates a TLR, therebyexposing tumor or infected cells to the host immune system imitating thesituation of a massive penetration through the intestinal wall. Theagent may be delivered systematically in solution for administrationsuch as intramuscularly. The agent may be a drug formulation thatexpresses the TLR agonist in the form of a nano-particle, which maycarry a functional agonist to the cell surface of a mammalian cell.

The agent may be a pharmaceutical agent comprising the drug formulationdescribed above, which may be produced using methods well known in theart. The agent may also comprise a coagent.

The vector may comprise a nucleic acid encoding flagellin. The vectormay be capable of expressing flagellin using a strong promoter. Theexpression vector may further comprise a leader sequence cloned upstreamof the gene encoding the TLR agonist. The drug formulation may be anadenovirus expressing:

the TLR agonist, delivered systematically in solution foradministration, such as intramuscularly; or

the TLR agonist, expressed in the form of nano-particles carryingfunctional TLR agonist, such as flagellin, which may be derived fromCBLB502, on their surface. The nano-particle may be on the basis of abacteriophage T7, or fully formed to retain its biological activity. Thenano-formulation may provide for dose-dependent, NF-κB-responsivereporter activation, and may result in cell internalization byendocytosis for effective immunization approach (Mobian AP-A).

a. Administration

Administration of the agents using the method described herein may besystemically, orally, parenterally, sublingually, transdermally,rectally, transmucosally, topically, via inhalation, via buccaladministration, or combinations thereof. Parenteral administrationincludes, but is not limited to, intravenous, intraarterial,intraperitoneal, subcutaneous, intramuscular, intrathecal, andintraarticular. Administration may also be subcutaneous, intravenous,via intra-air duct, or intra-tumoral. For veterinary use, the agent maybe administered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal. The agents may be administered to a human patient,cat, dog, large animal, or an avian.

The agent may be administered as a monotherapy or simultaneously ormetronomically with other treatments, which may be a surgery or removalof a tumor. The term “simultaneous” or “simultaneously” as used herein,means that the agent and other treatment be administered within 48hours, preferably 24 hours, more preferably 12 hours, yet morepreferably 6 hours, and most preferably 3 hours or less, of each other.The term “metronomically” as used herein means the administration of theagent at times different from the other treatment and at a certainfrequency relative to repeat administration.

The agent may be administered at any point prior to another treatmentincluding about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr,106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr,6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. The agentmay be administered at any point prior to a second treatment of theagent including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr,108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins., 10 mins., 9mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and1 mins.

The agent may be administered at any point after another treatmentincluding about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr,24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104hr, 106 hr, 108 hr, 110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hr.The agent may be administered at any point prior after a secondtreatment of the agent including about 120 hr, 118 hr, 116 hr, 114 hr,112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50mins., 45 mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins., 15mins., 10 mins., 9 mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3mins, 2 mins, and 1 mins.

b. Formulation

The method may comprise administering the agent. Agents provided hereinmay be in the form of tablets or lozenges formulated in a conventionalmanner. For example, tablets and capsules for oral administration maycontain conventional excipients may be binding agents, fillers,lubricants, disintegrants and wetting agents. Binding agents include,but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth,mucilage of starch and polyvinylpyrrolidone. Fillers may be lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants may be potato starch and sodium starch glycollate. Wettingagents may be sodium lauryl sulfate. Tablets may be coated according tomethods well known in the art.

Agents provided herein may also be liquid formulations such as aqueousor oily suspensions, solutions, emulsions, syrups, and elixirs. Theagents may also be formulated as a dry product for constitution withwater or other suitable vehicle before use. Such liquid preparations maycontain additives such as suspending agents, emulsifying agents,nonaqueous vehicles and preservatives. Suspending agent may be sorbitolsyrup, methyl cellulose, glucose/sugar syrup, gelatin,hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel,and hydrogenated edible fats. Emulsifying agents may be lecithin,sorbitan monooleate, and acacia. Nonaqueous vehicles may be edible oils,almond oil, fractionated coconut oil, oily esters, propylene glycol, andethyl alcohol. Preservatives may be methyl or propyl p-hydroxybenzoateand sorbic acid.

Agents provided herein may also be formulated as suppositories, whichmay contain suppository bases such as cocoa butter or glycerides. Agentsprovided herein may also be formulated for inhalation, which may be in aform such as a solution, suspension, or emulsion that may beadministered as a dry powder or in the form of an aerosol using apropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Agents provided herein may also be formulated as transdermalformulations comprising aqueous or nonaqueous vehicles such as creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

Agents provided herein may also be formulated for parenteraladministration such as by injection, intratumor injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The agent may also be provided in apowder form for reconstitution with a suitable vehicle including, butnot limited to, sterile, pyrogen-free water.

Agents provided herein may also be formulated as a depot preparation,which may be administered by implantation or by intramuscular injection.The agents may be formulated with suitable polymeric or hydrophobicmaterials (as an emulsion in an acceptable oil, for example), ionexchange resins, or as sparingly soluble derivatives (as a sparinglysoluble salt, for example).

c. Dosage

The method may comprise administering a therapeutically effective amountof the agent to a patient in need thereof. The therapeutically effectiveamount required for use in therapy varies with the nature of thecondition being treated, the length of time desired to activate TLRactivity, and the age/condition of the patient. In general, however,doses employed for adult human treatment typically are in the range of0.001 mg/kg to about 200 mg/kg per day. The dose may be about 1 mg/kg toabout 100 mg/kg per day. The desired dose may be convenientlyadministered in a single dose, or as multiple doses administered atappropriate intervals, for example as two, three, four or more sub-dosesper day. Multiple doses may be desired, or required.

The dosage may be at any dosage such as about 0.1 mg/kg, 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg,1 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 425 mg/kg, 450 mg/kg, 475 mg/kg,500 mg/kg, 525 mg/kg, 550 mg/kg, 575 mg/kg, 600 mg/kg, 625 mg/kg, 650mg/kg, 675 mg/kg, 700 mg/kg, 725 mg/kg, 750 mg/kg, 775 mg/kg, 800 mg/kg,825 mg/kg, 850 mg/kg, 875 mg/kg, 900 mg/kg, 925 mg/kg, 950 mg/kg, 975mg/kg or 1 mg/kg.

d. Monotherapy

The agent may be administered as a monotherapy, under which the agent isnot administered together with any other type of cancer treatment, suchas chemotherapy, radiation therapy, another biological therapy, or othercombination therapies; provided that “monotherapy” may includeadministration of the agent together with surgical treatment. The agentmay be administered in combination with a surgery, which may be tumorremoval. The agent may be administered prior to, together with, or afterthe surgery. The agent may be administered during the surgery.

4. METHOD FOR TREATING CANCER

Provided herein is a method for treating cancer, which may be present ina tissue that expresses a TLR such as TLR5, by administering to a mammalin need thereof the agent. The cancer may be a tumor or a metastaticcancer. The cancer may also be present in liver, bladder, lung, orintestinal tissue, and also may have originated in another type oftissue such as colon, breast, or prostate. The cancer may also bemelanoma or a hematological malignancy such as lymphoma. The cancer mayalso be any cancer that has metastasized to a TLR-expressing tissue,such as liver, lung, bladder, intestine, or other TLR-expressing tissue.The cancer may be a TLR-negative cancer, and thus lack expression of aToll-Like Receptor. The cancer may lack both endogenous and exogenousexpression of the Toll-Like Receptor. The method may comprise a step ofnot providing the Toll-Like Receptor to the cancer, which may includenot providing the Toll-Like Receptor either exogenously or endogenously.The cancer may lack any and all Toll-Like Receptor expression.

a. Toll-Like Receptor

The Toll-Like Receptor (TLR) may recognize molecules that are conservedmolecular products derived from pathogens that include Gram-positive,Gram-negative bacteria, fungi, and viruses, but are distinguishable fromhost molecules, collectively referred to as pathogen-associatedmolecular patterns (PAMPs). The TLR may also recognize endogenousmolecules released from injured or dying cells, collectively referred toas damage-associated molecular pattern (DAMPs). A PAMP or DAMP may be aTLR agonist as further described below. The TLR may be a fragment,variant, analog, homolog or derivative that recruits adapter moleculeswithin the cytoplasm of cells in order to propagate a signal. The TLRmay be from a human or other mammalian species such as rhesus monkey,mouse, or rat. The TLR may be at least 30-99% identical to a TLR thatrecruits adapter molecules within the cytoplasm of cells in order topropagate a signal.

The TLR may be one of the between ten and fifteen types of TLR that areestimated to exist in most mammalian species. The TLR may be one of the13 TLR (named simply TLR1 to TLR13) that have been identified in humansand mice together, or may be an equivalent form that has been found inother mammalian species. The TLR may be one of the 11 members(TLR1-TLR11) that have been identified in humans.

The TLR may ordinarily be expressed by different types of immune cells,and may be located on the cell surface or in the cell cytoplasm. The TLRmay ordinarily be expressed on cancer cells. The TLR may ordinarily beexpressed by normal epithelial cells in the digestive system, normalkeratinocytes in the skin, alveolar and bronchial epithelial cells, andepithelial cells of the female reproductive tract. These cells lining anorgan may be the first line of defense against invasion ofmicroorganisms, and TLRs ordinarily expressed in epithelial cells mayhave a crucial role in the regulation of proliferation and apoptosis.

The TLR may not be expressed by the cancer cells. The TLR-negativecancer cells may not express any TLR mRNA, may not express any TLRprotein, or may not express any functional TLR protein. The TLR proteinmay not function due to reduced ability to bind a TLR ligand or reducedability to transmit downstream signals triggered by ligand binding. TheTLR-negative cancer cells may also have reduced levels of TLR mRNA,protein, or TLR function. The reduction may be 100%, or by more than99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%, ascompared to a normal cell from the tissue from which the cancer celloriginated, or as compared to another, known TLR-expressing cell type.The TLR-expressing cell may be a normal cell or a tumor cell, such as atumor cell line or tumor xenograft.

The TLR ordinarily expressed on cancer cells may upregulate the NF-κBcascade and produce anti-apoptotic proteins that contribute tocarcinogenesis and cancer cell proliferation.

Four adapter molecules of TLRs are known to be involved in signaling.These proteins are known as myeloid differentiation factor 88 (MyD88),Tirap (also called Mal), Trif, and Tram. The adapters activate othermolecules within the cell, including certain protein kinases (IRAK1,IRAK4, TBK1, and IKKi) that amplify the signal, and ultimately lead tothe induction or suppression of genes that orchestrate the inflammatoryresponse. TLR signaling pathways during pathogen recognition may induceimmune reactions via extracellular and intracellular pathways mediatedby MyD88, nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB), and mitogen-associated protein kinase (MAPK). In all, thousandsof genes are activated by TLR signaling, and collectively, the TLRconstitute one of the most pleiotropic, yet tightly regulated gatewaysfor gene modulation.

TLRs together with the Interleukin-1 receptors form a receptorsuperfamily, known as the “Interleukin-1 Receptor/Toll-Like ReceptorSuperfamily.” All members of this family have in common a so-called TIR(Toll-IL-1 receptor) domain. Three subgroups of TIR domains may exist.Proteins with subgroup I TIR domains are receptors for interleukins thatare produced by macrophages, monocytes and dendritic cells and all haveextracellular Immunoglobulin (Ig) domains. Proteins with subgroup II TIRdomains are classical TLRs, and bind directly or indirectly to moleculesof microbial origin. A third subgroup of proteins containing TIR domains(III) consists of adaptor proteins that are exclusively cytosolic andmediate signaling from proteins of subgroups 1 and 2. The TLR may be afragment, variant, analog, homolog or derivative that retains either asubgroup I TIR domain, subgroup II TIR domain, or subgroup III TIRdomain.

The TLR may function as a dimer. For example, although most TLRs appearto function as homodimers, TLR2 forms heterodimers with TLR1 or TLR6,each dimer having a different ligand specificity. The TLR may alsodepend on other co-receptors for full ligand sensitivity, such as in thecase of TLR4's recognition of LPS, which requires MD-2. CD14 and LPSBinding Protein (LBP) are known to facilitate the presentation of LPS toMD-2.

(1) TLR1

The TLR may be TLR1, which recognizes PAMPs with a specificity forgram-positive bacteria. TLR1 has also been designated as CD281.

(2) TLR5

The TLR may be Toll-Like Receptor 5. The protein encoded by the TLR5 mayplay a fundamental role in pathogen recognition and activation of innateimmunity. TLR5 may recognize PAMPs that are expressed on infectiousagents, and mediate the production of cytokines necessary for thedevelopment of effective immunity. TLR5 may recognize bacterialflagellin, a principal component of bacterial flagella and a virulencefactor. The activation of the TLR5 may mobilize the nuclear factor NF-κBand stimulate tumor necrosis factor-alpha production.

(3) Cancer Type

The cancer may be a primary cancer or a metastatic cancer. The primarycancer may be an area of cancer cells at an originating site thatbecomes clinically detectable, and may be a primary tumor. In contrast,the metastatic cancer may be the spread of a disease from one organ orpart to another non-adjacent organ or part. The metastatic cancer may becaused by a cancer cell that acquires the ability to penetrate andinfiltrate surrounding normal tissues in a local area, forming a newtumor, which may be a local metastasis.

The metastatic cancer may also be caused by a cancer cell that acquiresthe ability to penetrate the walls of lymphatic and/or blood vessels,after which the cancer cell is able to circulate through the bloodstream(thereby being a circulating tumor cell) to other sites and tissues inthe body. The metastatic cancer may be due to a process such aslymphatic or hematogeneous spread. The metastatic cancer may also becaused by a tumor cell that comes to rest at another site, re-penetratesthrough the vessel or walls, continues to multiply, and eventually formsanother clinically detectable tumor. The metastatic cancer may be thisnew tumor, which may be a metastatic (or secondary) tumor.

The metastatic cancer may be caused by tumor cells that havemetastasized, which may be a secondary or metastatic tumor. The cells ofthe metastatic tumor may be like those in the original tumor. As anexample, if a breast cancer or colon cancer metastasizes to the liver,the secondary tumor, while present in the liver, is made up of abnormalbreast or colon cells, not of abnormal liver cells. The tumor in theliver may thus be a metastatic breast cancer or a metastatic coloncancer, not liver cancer.

The metastatic cancer may have an origin from any tissue. The metastaticcancer may originate from melanoma, colon, breast, or prostate, and thusmay be made up of cells that were originally skin, colon, breast, orprostate, respectively. The metastatic cancer may also be ahematological malignancy, which may be lymphoma. The metastatic cancermay invade a tissue such as liver, lung, bladder, or intestinal. Theinvaded tissue may express a TLR, while the metastatic cancer may or maynot express a TLR.

b. Combination

The method may also comprise co-administration of the TLR agonist withan anti-cancer therapy. The anti-cancer therapy may be FAS ligand, a FASagonistic antibody, TNFα, a TNFα agonistic antibody, TRAIL, or a TRAILagonistic antibody. The TLR5 agonist may be used to sensitize the cancerto the anti-cancer therapy. The method may also be combined with othermethods for treating cancer, including use of an immuno stimulant,cytokine, or chemotherapeutic. The immunostimulant may be a growthhormone, prolactin or vitamin D.

5. METHOD OF REDUCING CANCER RECURRENCE

Also provided herein is a method of reducing cancer recurrence,comprising administering to a mammal in need thereof a TLR agonist. Thecancer may be or may have been present in a tissue that either does ordoes not express TLR, such as TLR5. The cancer, tissue, TLR, mammal, andagent may be as described above. The method may also prevent cancerrecurrence. The cancer may be an oncological disease.

The cancer may be a dormant tumor, which may result from the metastasisof a cancer. The dormant tumor may also be left over from surgicalremoval of a tumor. The cancer recurrence may be tumor regrowth, a lungmetastasis, or a liver metastasis.

6. MAMMAL

The mammal may have a fully-functional immune system, and may not beimmunocompromised. The mammal may also have a level of immunity that isequivalent to the level sufficient to make the mammal eligible for afirst or second round a chemotherapy. The mammal may not have a lowwhite blood cell count, which may be chemotherapy-induced. The low whiteblood cell count may be caused by the loss of healthy cells duringchemotherapy. The loss may be an expected side effect of a chemotherapydrug. The low white blood cell count may be a severe immunosuppressioncaused by chemotherapy. The low white blood cell count may compromisethe antitumor effect of the agent. The low white blood cell count may berestored 7-14 days after a chemotherapy treatment.

The mammal may have a white blood cell count that is within a normalrange. The mammal may also have a white blood cell count that isindicative of mild immunosuppression. The mammal may have not receivedchemotherapy treatment for 7-14 days, or at least 14 days. The mammalmay also have total white blood cell count of at least 3000 or 3500cells/ml of whole blood; a granulocyte count of at least 1800 or 2100cells/ml of whole blood; or an albumin level of at least 3.0 or 3.5g/100 ml of whole blood. The white blood cell count, granulocyte count,or albumin level may also fall within +/−5%, 10%, 20%, 30%, 40%, or 50%of these levels.

7. METHOD OF PROTECTING LIVER

As discussed above, anti-cancer treatments that trigger apoptosisthrough FAS, TRAIL, and TNFα death receptor signaling, such as deathligands, can cause severe liver toxicity. Thus, the use of moleculessuch as FAS, TRAIL, and TNFα as anti-cancer treatments has been limited,despite the efficacy of these molecules in targeting cancer cells.Accordingly, also provided herein is a method of protecting liver tissuein a mammal from the effects of a liver toxicity. The liver may beprotected by administering the agent to the mammal. The death receptorsignaling agonist may be FAS, TRAIL, or TNFα. The death ligand may be aliver toxicity. The FAS, TRAIL, or TNFα may be used as an anti-canceragent.

The liver toxicity may also be a Salmonella infection, which may be fromSalmonella typhimurium. The agent may also be used to protect againstliver toxicity that may be FAS-mediated. The toxicity may also be FASligand, a FAS agonistic antibody, TNFα, acetaminophen, alcohol, a viralinfection of the liver, or a chemotherapeutic agent. The agent may beadministered to the mammal.

Example 1 An Agonist of TLR5 Protects Liver from Hepatotoxicity

CBLB502, which is a pharmacologically optimized TLR5 agonist, is apowerful radioprotectant due to, at least in part, inhibition ofapoptosis in radiosensitive tissues. CBLB502 was tested for liverprotection from Fas-mediated apoptosis. The following examplesdemonstrate that upon stimulation with CBLB502 the TLR5 pathway isactive in liver hepatocytes of mice and humans leading toNF-kB-dependent induction of genes encoding anti-apoptotic proteins.Pretreatment of mice with CBLB502 protected them from lethal doses ofFas agonistic antibodies, reduced Fas-induced elevation of liver enzymesin the blood, caspase activity in liver extracts and preserved livertissue integrity. CBLB502 did not protect tumors in syngeneic melanomaand colon carcinoma mouse models. These observations support the use ofFas agonists for cancer treatment under the protection of a TLR5agonist, such as CBLB502.

NF-kB response was compared in different organs after administration ofTLR5 agonist CBLB502 and TLR4 agonist LPS, another known activator ofNF-kB. CBLB502 was found to induce fast direct activation of NF-kB inhepatocytes, while LPS activation of NF-kB in hepatocytes was mediatedthrough different types of cells. The following data thus alsodemonstrate that pre-treatment with CBLB502 can reduce Fas-mediatedhepatotoxicity during anti-cancer therapy in mice. The approachesdescribed below are based on the increasing the resistance of normaltissues to damaging side effects through activation of NF-kB signalingby toll-like receptor-5 (TLR5) agonist CBLB502 derived from flagellin ofSalmonella typhimurium.

1. Determination of NF-k Activation In Vivo in Response to TLR4 and TLR5Agonists.

NF-kB response was investigated in different organs of mice to TLR5agonist CBLB502 in comparison with bacterial LPS acting through TLR4.NF-kB dependent luciferase reporter Xenogen mouse model in whichluciferase transgene is expressed under the control of NFkB-dependentnatural promoter of IkBα gene (Zhang N, et al, 2005). Uponadministration of NFkB-activating agents, luciferase activity wasincreased in cells and tissues that respond to a given agent. Usingnoninvasive Xenogen imaging system and ex vivo luciferase reporterassay, detected strong activation of NF-kB in liver of mice was detected2 hours after s.c. injection of CBLB502 (FIG. 7A). The quantitativeanalysis of NF-kB activation in different organs revealed that incomparison with LPS, CBLB502 induced much stronger activation of NF-kBin liver, similar high NF-kB activation level in the intestine, whileless NF-kB activity was found in spleen, bone marrow, kidney and lungs(FIG. 7B). The dynamics of NF-kB induced luciferase reporter activitywas similar for both TLR agonists with the activation profile peakingapproximately two hours after injection, reduced at the six hour timepoint and effectively undetectable 24 hours post-injection (FIG. 10).

Immunohistochemical staining of mouse liver samples for p65translocation to the nuclei revealed that CBLB502 directly activatedNF-kB in hepatocytes as early as 20 min after injection with no responseof Kupffer and endothelial cells yet (FIG. 7C). By 1 h after CBLB502injection, all liver cells including Kupffer cells and endothelium cellsdemonstrated nuclear accumulation of p65 suggesting overlap of primaryand secondary effects with subsequent activation of NF-kB by paracrinemechanisms. In contrast, LPS-activated NF-kB nuclear translocation inhepatocytes occurred significantly later. The activation of NF-kB wasobserved first in Kupffer and endothelial cells followed by theengagement of hepatocytes about 1 h after LPS administration.

Primary hepatocyte cultures (murine and human) treated with CBLB502, butnot with LPS, demonstrated NF-kB translocation to the nuclei (FIG. 7D,E). CBLB502 mediated NF-kB activation was confirmed by NF-kB dependentluciferase expression with murine hepatocyte cell culture, while LPS didnot induce NF-kB activation in this cells (FIG. 11). Small level ofNF-kB activation found in LPS-treated hepatocytes was more likely due tocontamination of primary hepatocyte culture with other stromal livercells.

These results show that hepatocytes express TLR5 but not TLR4 allowingCBLB502 to directly activate NF-kB in hepatocytes while LPS initiallyactivates other cell types (immune and/or stromal) and only laterindirectly activates hepatocytes as a secondary event.

2. CBLB502 Protection from Fas Mediated Hepatotoxicity

As it has been demonstrated, the anti-Fas antibodies can inducedose-dependent hepatotoxicity and rapidly kill mice by inducingapoptosis, liver tissue necrosis and hemorrhage (Ogasawara J et al,Nature 1993, Nishimura et al 1997). Thus, NF-kB activation inhepatocytes induced by TLR5 agonist CBLB502 may protect liver from Fasmediated apoptosis. In NIH-Swiss mice, 4 μg of anti-Fas antibodies(clone Jo2) injected i.p. induced massive apoptosis, necrosis andhemorrhage in liver (FIGS. 8B, C and D) killing mice within first 1-2days after antibody injections (FIG. 8A). Pathomorphological examinationof CBLB502-treated mice in dynamics compared to intact control miceshowed that their livers had slight vacuolization of the hepatocytes(FIG. 12). The examination of mice injected with sub-lethal dose ofanti-Fas antibodies (3 μg/mouse) in dynamics revealed pronouncedapoptosis of the hepatocytes around the portal tracts with betterpreserved cells adjacent to the terminal (central) venues, mostpronounced at 5 hrs and diminishing with time (12 and 24 hourspost-injection). In the livers of mice treated with CBLB502 and anti-Fasantibodies the changes were minimal and the hepatocytes looked close tonormal—only slight vacuolization and single apoptotic cells werevisible.

CBLB502 injected mice had much less damage to the liver that deflectedin better overall survival after injections of about than 80% ofNIH-Swiss mice when injected 30 min before anti-Fas antibodies (FIG.8A). All mice survived when CBLB502 was injected 2 hours beforeantibodies. The protection level then declined by 6 hours time-point ofpre-treatment.

Two and three μg of anti-Fas antibodies induced only transient livertoxicity in NIH-Swiss mice, caspase 3/7 activation in the liver andalanine aminotransferase (ALT) secretion in the blood (FIG. 8E, F). Bothtests showed significant reduction of liver damage induced by anti-Fasantibodies if mice were pre-treated with CBLB502. Interestingly, Balb/cand C57B1/6 mice appeared to be less sensitive to anti-Fas antibodiesthan NIH-Swiss mice. Four μg of anti-Fas antibodies, the lethal dose forNIH-Swiss mice, induced only transient caspase 3/7 activation in BALB/cand C57Bl/6 mice which was successfully prevented by CBLB502 injection30 min before antibodies (FIG. 13).

These data support the hypothesis that TLR5 mediated NF-kB activation inhepatocytes can be an indicator and a measure of increased resistance toFas-mediated toxicity.

3. Suppression of pro-apoptotic and induction of anti-apoptotic factorsby CBLB502 in liver.

Caspases 3 and 7 are downstream targets of both intrinsic(mitochondrial) and extrinsic (caspase) Fas-mediated apoptosissignaling. Upon activation of the receptor, first caspase-8 becomesphosphorylated and cleaved leading to activation of mitochondrialapoptotic mechanism acting through cleavage of pro-apoptotic Bid proteinand cytochrome release (Lou et al 1998). Therefore we examined whetherCBLB502 suppresses this mechanism.

Western blot analysis of liver protein extracts for both caspases-8 andBid demonstrated much less cleavage of these proteins in mice injectedwith combination of CBLB502 and anti-Fas antibodies in comparison with asingle injection of anti-Fas antibodies (FIG. 9A, B). Consistently,caspase 8 activation was reduced to a background level, as indicated byusing fluorigenic substrate assay (FIG. 8F).

The fact that the protection of mice from Fas-mediated hepatotoxicity byCBLB502 is increased with time with maximum peaking at 30 min-2 hourssuggests the existing of pre-conditioning events in hepatocytes. Amongthe numerous of cytokines and anti-apoptotic factors, the up-regulationof two anti-apoptotic bcl2 family members bcl2A1B and bcl2A1D (Chao andKorsmeyer, 1998, Arikawa et al 2006) was found in livers by RNA arrayhybridization 30 min and 2 hours after CBLB502 administration that wasconfirmed by RT-PCR (FIG. 9C). CBLB502 also quickly induced RNAexpression of another anti-apoptotic protein immediate early responseprotein IER-3 (FIG. 9C, IEX-1 is an alternative name) that was shownsuppressing the production of reactive oxygen species and mitochondrialapoptotic pathway (Shen et al 2009). RT-PCR analysis of liver samplesrevealed the induction of IER-3 RNA expression by CBLB502 already 30 minafter administration with significant increase by 2 hours. Severalproteins of MAPK pathway were found up-regulated in livers of CBLB502treated mice. It was demonstrated that activation of MAPK pathway intumors mediates the resistance of these cells to Fas receptor apoptosis(REF). The up-regulation of Jun, Jun-B and Fos gene expressions directlycorrelated with mouse survival after anti-Fas antibody injectionsfollowed the pre-treatment with CBLB502 suggesting their possible rolein CBLB502 mediated protection from Fas hepatotoxicity.

4. Effect of CBLB502 on Fas-Mediated Antitumor Activity

LPS is not a good candidate for clinical application, since it inducesstrong inflammation in many organs and can be directly cytotoxic throughFADD/caspase-8 apoptotic pathway (REFs). CBLB502 in its turn has beentested in mice, non-human primates and human healthy volunteers andfound to be a rather mild inducer of short-lasting inflammation. Whenevaluating a tissue protecting compounds, there is always possibilitythat by reducing toxic side effects it can also make tumor cells moreresistant and jeopardize the efficacy of antitumor therapy. The in vivoantitumor effect of combination treatment with CBLB502 and anti-Fasantibodies was tested in CT-26 colon carcinoma mouse model of s.c.growing tumors and experimental liver metastases. This tumor model wasused in a recently published study applying FasL-expressing S.typhimurium, total attenuated bacteria, to deliver FasL to the tropictumors and to induce Fas mediated antitumor effect (Loeffler et al2008). CT-26 tumor cells and A20 lymphoma cells do not express TLR5, asdetermined by RT-PCR and a NF-kB dependent luciferase reporter assay(FIG. 14). Here, tumor-bearing mice were treated with anti-Fasantibodies alone or combination of recombinant CBLB502 given twice 24 hsand 1 h before a single injection of anti-Fas antibodies (4 μg/mouse,FIG. 9D). The volumes of s.c. growing tumors in treated mice werecompared with tumors growing in the intact mice. CT-26 tumors were foundto be rather resistant to the toxic but not lethal dose of anti-Fasantibodies (FIG. 9D). Pre-treatment with CBLB502 slightly sensitizedtumors to anti-Fas antibodies reflecting in growth-inhibitory tumorresponse. Fas mediated antitumor effect was tested in the experimentalmodel of liver metastases induced by intrasplenic injection ofluciferase expressing CT-26 tumor cells followed by splenectomy. Hepatictumor growth was assessed using Xenogen luciferase imaging every 4-6days after the treatment. Mice remained free from liver tumor growthwere counted at each imaging procedure (FIG. 9E). The resultsdemonstrate significant delay of tumor appearance (FIG. 9G) and growthin livers by both treatments, anti-Fas antibody alone or given afterpre-treatment with CBLB502. The increased sensitivity of TLR5 negativeCT-26 tumors to combination treatment with anti-Fas and CBLB502 suggeststhe activation of antitumor immune response against CT-26 tumors.Indeed, the immunohistochemical analysis of liver sample with CT-26tumors taken 24 hours after anti-Fas/CBLB502 treatment revealed theaccumulation of neutrophils in inside and around of tumor nodules (FIG.9F). Thus, CBLB502 does not protect tumors from anti-Fas antibodiestoxicity and can even slightly enhance Fas mediated antitumor effectagainst CT-26 tumors. The simultaneous protection of normal liver tissuefrom Fas mediated toxicity may allow increasing the amount of the Fasagonist reaching complete prevention of liver metastases and thetherapeutic effect against s.c. growing tumors.

Materials and Methods

Mice

NIH-Swiss female mice were purchased from NCI (Frederick, Md.), BALB/cand C57Bl/6 female mice were purchased from Jackson Laboratory (BarHarbor, Me.). All mice were used in the experiments at the age of 10-14weeks old. Balb/C-Tg (IκBα-luc)Xen mice with NF-kB inducible luciferasereporter gene were originally purchased from Xenogen (Alameda, Calif.)and bred in our domestic colony.

Reagents

CBLB502, a bacterial flagellin derivative, was obtained from ClevelandBioLabs, Inc. Bacterial lipopolysacharide (LPS) from Escherichia coli055:B5 was purchased from Sigma. Purified agonistic hamster anti-mouseFas antibodies, clone Jo2, were purchased from BD Biosciences.

Analysis of NF-κB Activation In Vivo Using NF-kB Reporter Mouse Model

BALB/c-Tg (IκBα-luc)Xen reporter mice were injected s.c. with CBLB502(0.2 mg/kg). The induction of NF-kB by CBLB502 was detected bynoninvasive in vivo imaging 2 hours after the treatment (FIG. 1A). Micewere injected with D-luciferin (3 mg/100 μl, i.p., Promega), immediatelyanesthetized with isofluorane and images were taken using Xenogen IVISImaging System 100 series. To quantify the results, samples of liver,lungs, kidney, spleen, heart and intestine from NF-kB reporter miceinjected s.c. with 100 μl of either PBS, CBLB502 (0.2 mg/kg) or LPS (1mg/kg) were obtained 2, 6 and 24 h after injections (FIG. 7B, 10).Tissue samples were covered with lysis buffer containing proteinaseinhibitor cocktail (according to manufacture's recommendation,Calbiochem) to get 100 mg tissue per 1 ml lysis buffer. This wasfollowed by homogenization and centrifugation at 14,000 rpm for 10 minat 4 C. Luciferase activity was measured in 20 μl of samples immediatelyafter adding 30 μl of luciferin reagent (Bright-Glo Luciferase AssaySystem, Promega). Luciferase activity was normalized per g of theprotein extract. Luciferase fold induction was calculated as ratiobetween average luciferase units in livers of the TLR ligand treatedmice and that obtained from PBS injected control mice.

Immunohistochemical Staining for p65 Translocation.

P65 localization was detected in livers isolated from NIH-Swiss miceinjected s.c either with CBLB502 (0.04 mg/kg) or LPS (1 mg/kg). Controlmice were injected with PBS. Tissue samples were obtained 20, 40 and 60min after the treatments, processed into paraffin blocks. All livertissues were stained with rabbit polyclonal antibody against NF-kB p65and rat monoclonal antibody against cytokeratin 8 followed byappropriate secondary fluorochrome-conjugated antibodies (p65—green,cytokeratin-8—red). The same staining was performed on the plates withprimary mouse hepatocytes isolated from EGTA (0.5 mM in PBS) perfusedliver tissues of NIH-Swiss mice followed by collagenase digestion andwith human hepatocyte culture purchased from (BD Biosciences). Bothtypes of hepatocytes were treated in vitro with CBLB502 (100 ng/ml) orLPS (1 μg/ml) for indicated period of time. Control hepatocytes remainedintact. Pictures were taken at ×20 magnification (FIG. 7C, D, E).

Survival Assay

NIH-Swiss mice were injected i.p. with 2, 3, 4, and 5 μg of anti-Fasantibodies in 200 μl of PBS to determine a 100% lethal dose that wasfound to be 4 μg/mouse for this mouse strain. Then CBLB502 (0.04 mg/kg,s.c.) was injected s.c. 30 min, 2 hours and 6 hours before 4 μg ofanti-Fas antibodies (i.p.) (FIG. 8A). Usually death from anti-Fashepatotoxicity occurs during first 1-2 days after antibody injections.Mouse survival was observed and recorded during 30 days.

TUNEL Staining of Apoptotic Cells in Liver

Apoptosis in the liver of NIH-Swiss mice five hours after injectionswith CBLB502 (s.c., 0.04 mg/kg) or PBS 30 min before anti-Fas antibodieswas detected in paraffin-embedded specimens. Apoptotic cells werestained by the indirect terminal deoxynucleotidyl transferase mediateddeoxyuridine tri-phosphate nick end labeling (TUNEL) method with TUNELPOD kit (Roche Applied Science) (FIG. 8B).

Histological Assessment of Liver Morphology

Liver specimens were collected from NIH-Swiss mice five hours (FIG. 2C)or in dynamics of 5, 12 and 26 hours (FIG. S3) after anti-Fas antibodyinjections with or without pre-treatment with CBLB502 (0.04 mg/kg) 30minutes before antibodies. Mice that were not treated (“intact”) wereused as controls. Tissue specimens were fixed in 10% buffered formalin,embedded in paraffin, sectioned and processed with H&E staining.

Histological Staining of Liver for Hemorrhage

Paraffin sections were stained with antibody against mouse IgGconjugated with Cy5 [Jackson Immunoresearch, pseudo-colored in purple]and mounted with ProLong Gold anti-fade reagent with DAPI[Invitrogen,blue nuclear stain]. Erythrocytes were fisualized in red channel by redautofluorescence. (FIG. 2D). Images were captured under Axiolmager Z1fluorescent microscope (Zeiss) equipped with AxioCam HRc 13 megapixeldigital camera using Axio Vision software (rel. 4.6.3)

Caspase Activation

Livers were cut to small pieces and homogenized with a tissue grinder(Bullet Blender, NextAdvance) in the buffer (10 mM Hepes, 0.4 mM EDTA,0.2% CHAPS, 2% glycerol), supplemented with 2 mM DTT. All steps wereperformed on ice. Liver homogenates were centrifuged for 20 min at13,000×g, and supernatant was stored at −20° C. Caspase activities weredetermined by incubation of liver homogenate (containing 50 μg of totalprotein) with 50 μM of the fluorogenic substrateacetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin (Ac-DEVD-amc)(ENZO, LifeSciences) in 200 μl cell-free system buffer containing 10 mMHEPES, 0.4 mM EDTA, 0.2% CHAPS, 2% glycerol and 2 mM DTT. The release offluorescent amc was measured after at time 0 and 2 hours of incubationat 37° C. by fluorometry (Ex: 355, Em: 485) (Victor3, PerkinElmer). Dataare shown as the difference between twp and zero hours (FIG. 8E).

Detection of Alanine-Aminotransferase (ALT) in the Serum of Anti-FasAntibody-Treated Mice with and without CBLB502 Injections

NIH-Swiss mice (3 per group) were injected s.c. with 1 μg CBLB502 30 minbefore anti-Fas antibodies. The alanine aminotransferase (ALT) presencein mouse serum was determined using commercial enzyme assays accordingto the manufacturer's instructions (Stanbio Laboratory, Boerne, Tex.,USA). Absorbance at 340 nm was measured at 60 second interval(ΔA/minute). (FIG. 8F)

Western Blot Analysis

Total protein was isolated from treated and untreated mouse liver usingRIPA buffer (Sigma-Aldrich St. Louis, Mo.) supplemented with proteaseinhibitor cocktail (Sigma-Aldrich St. Louis, Mo.). The protein extractswere separated by electrophoresis in denaturing 4 to 20% polyacrylamideNovex gels (Invitrogen, Carlsbad, Calif.) and transferred to nylonpolyvinylidene difluoride (PVDF) membranes (Immobilon-P, MilliporeBillerica Mass.). The following antibodies were used: Caspase-8 antibody(Calbiochem, Darmstadt, Germany), anti-BID (AbCam, Cambridge Mass.).Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit andanti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, Calif.). (FIGS. 9A and 9B)

RNA Analysis

Total RNA was extracted from treated and untreated mouse livers usingTRIzol reagent according to manufacturer instructions (Invitrogen,Carlsbad, Calif.). To eliminate any eventual contamination with genomicDNA, isolated RNAs were treated with DNaseI (Invitrogen, Carlsbad,Calif.). cDNAs were synthesized by using SuperScript™ II ReverseTranscriptase and oligo(dT)12-18 primer (Invitrogen, Carlsbad, Calif.),according to manufacturer instructions. RNA expression of Bcl2A1B,Bcl2A1D, IER-3, Fos, Jun and JunB genes in livers of intact mice andtreated with CBLB502 and LPS for 30 min and 2 hours was detected byRT-PCR. GAPDH was used as a control to monitor the induction of geneexpression. The primers were designed using LaserGene software (DNASTAR,Inc., Madison, Wis.) and then UCSC Genome Browser In-Silico PCR websitewas used to check for locating primers. Primers specific for the IER3gene (GenBank Accession No. NM_(—)133662.2) (sense5′-ACTCGCGCAACCATCTCCACAC-3′ and antisense5′-CTCGCACCAGGTACCCATCCAT-3′), Bcl2A1B gene (GenBank Accession No.NM_(—)007534.3) (sense 5′-TAGGTGGGCAGCAGCAGTCA-3′ and antisense5′-CTCCATTCCGCCGTATCCAT-3′), Bcl2A1D gene (GenBank Accession No.NM_(—)007536.2) (sense 5′-TCTAGGTGGGCAGCAGCAGTC-3′ and antisense5′-ATTCCGCCGTATCCATTCTCC-3′), Jun (GenBank Accession No. NM_(—)010591.2)(sense 5′-TGAAGCCAAGGGTACACAAGAT-3′ and antisense5′-GGACACCCAAACAAACAAACAT-3′), Fos (GenBank Accession No.NM_(—)010234.2) (sense 5′-GAGCGCAGAGCATCGGCAGAAG-3′ and antisense5′-TTGAGAAGGGGCAGGGTGAAGG-3′), JunB (GenBank Accession No.NM_(—)008416.2) (sense 5′-AGCCCTGGCAGCCTGTCTCTAC-3′ and antisense5′-GTGATCACGCCGTTGCTGTTGG-3′) and GAPDH gene (sense5′-ACCACAGTCCATGCCATCAC-3′ and antisense 5′-TCCACCACCATGTTGCTGTA-3′)were used. Amplification of cDNA was done for 20-30 cycles usingspecific primer pairs for each gene (FIG. 3C).

Experimental Therapy of CT-26 Tumor-Bearing Mice

The effect of CBLB502 on the sensitivity of tumors to anti-Fasantibodies was analyzed using two models of syngenic colonadenocarcinoma CT-26 tumor: 1) CT-26 s.c. growing tumors, and 2)Experimental liver metastatic model of CT-26 tumors. CT-26 cells weretransduced with lentiviral vector carrying luciferase gene under CMVpromoter for constitutive expression of luciferase. Tumors were inducedby s.c. injections of CT-26 tumor cells (2.5×105/100 μl) in both flanksof BALB/c mice. When the tumors reached about 4-5 mm in diameter, themice were randomly divided into three groups and treatment wasinitiated. One group of mice was injected i.p. with anti-Fas antibodies(4 μg/mouse), another was treated with CBLB502 (1 μg/mouse) 24 h and 1 hbefore anti-Fas antibody injection (4 μg/mouse). Control mice (‘intact’)received PBS injections s.c. and i.p. in replace of CBLB502 andantibodies. Tumor volumes were measured every second day using calipersand calculated by formula: V=Π/6*a2*b, where a<b. Survival was followedfor 2 weeks when experiment was terminated due to large tumors in thecontrol group (FIG. 9D). Statistical difference between tumor volumeswas estimated using ANOVA one-way analysis of variances (p<0.05). Forthe development of liver tumor growth, CT-26 tumor cells (2×105/50 μl)were injected directly into spleen followed by splenectomy 5 min later.Mice were treated with anti-Fas antibodies and combination of CBLB502with antibodies the same way as described for s.c. tumors starting onday 5 after tumor cell inoculation. Noninvasive bioluminescent imagingof mice anesthetized with isoflurane and injected with D-luciferin (3mg/100 μl, i.p.) was performed using Xenogen IVIS Imaging System 100series on the days 14, 17, 22 and 28 after tumor cell injection. Micewere sacrificed when tumor growth in liver was determined. Statisticalcomparison of liver tumor-free curves was done using log-rank(Mantel-Cox) test (p<0.05) (FIG. 9G).

Example 2

Antitumor activity of CBLB502 on colon HCT116 adenocarcinoma s.c. growthin xenogenic model of athymic mice. HCT116 were injected s.c. into 2flanks of 8 athymic nude mice (0.5×106/100 μl of PBS) to induce tumors.When tumors became of about 3-5 mm in diameter (by day 6 afterinjections) mice were randomly distributed into 2 groups, 5 mice forCBLB502 treated group and 3 mice in PBS control group. Suppression oftumor growth was determined in CBLB502 treated mice. Data are shown inFIG. 15.

Example 3

Antitumor activity of CBLB502 on 293-TLR5 s.c. tumor growth in xenogenicmodel of athymic mice. Tumor cells were injected s.c. into 2 flanks of10 athymic nude mice (2×10⁶/100 μl of PBS) to induce tumors. When tumorsbecame of about 3-5 mm in diameter (by day 7 after injections) mice wererandomly distributed into 2 groups, 5 mice for CBLB502 treated group and5 mice in PBS control group. Suppression of tumor growth was found inCBLB502 treated mice. Data are shown in FIG. 16.

Example 4

Antitumor activity of CBLB502 on A549 adenocarcinoma s.c. growth inxenogenic model of athymic mice. The original A549 cells (ATCC, CLL-185)were injected s.c. into 2 flanks of 8 athymic nude mice (0.5×10⁶/100 μlof PBS) to induce tumors. When tumors became of about 3-5 mm in diameter(by day 6 after injections) mice were randomly distributed into 2groups, 5 mice for CBLB502 treated group and 3 mice in PBS controlgroup. A549 tumor-bearing mice were injected with either CBLB502 (1μg/mouse) or PBS three times with a 24-hr time interval. In the PBSinjected control group of mice, tumor volumes gradually and regularlyincreased. On the other hand, the CBLB502 injected mice expressedinhibited tumor growth during the first several days after injectionsand then tumor growth restored. The second round of CBLB502 injections 2weeks after the first treatment (days 14, 15 and 16) induced analogoustumor growth inhibition for approximately 1-2 weeks before the restartof tumor growth. As a result, by the end of the experiment the sizes ofthe A549 tumors differed significantly in the two groups of mice, beingmuch smaller in CBLB502 treated vs. PBS treated mice. Data are shown inFIG. 17.

Example 5

Antitumor effect of CBLB502 on syngenic orthotopically (s.c.) growingsquamous cell carcinoma SCCVII tumors. The rate of SCCVII orthotopictumor growth in syngenic C3H mice after CBLB502 or PBS (no treatment)treatments (0.1 mg/kg, s.c. days 1, 2, 3) to reach 400 mm³ tumor size,n=6-10. The x-axis in FIG. 18 represents the amount of days needed fortumors to reach 400 mm³ volume with and without treatment with CBLB502.Data are shown in FIG. 18.

Example 6

Antitumor activity of CBLB502 in Fischer rats bearing s.c. advanced Wardcolorectal carcinoma. CBLB-502 was administered by i.p. once a day for 5days (0.2 mg/kg×5 doses) initiated 5 days after tumor transplantationinto 4 rats. Control 4 rats received PBS injection as a vehicle control.Tumor weight was measured daily. Complete response (tumor completedisappearance) was observed in 3 rats treated with CBLB502 (FIG. 19).The fourth rat in this group had tumor growth similar to rats in thecontrol group.

Example 7

The effect of CBLB502 injections on A549 tumors differing in TLR5expression (A549-shTLR5 vs. A549-shV). In order to suppress TLR5expression, A549 cells expressing Firefly luciferase gene under thecontrol of NF-kB promoter (Cellecta, Mountain View, Calif.) weretransduced with lentiviral pLKO1-puro vector expressing shRNA specificto human TLR5 gene[CCG-GCC-TTG-CCT-ACA-ACA-AGA-TAA-ACT-CGA-GTT-TAT-CTT-GTT-GTA-GGC-AAG-GTT-TTT-G]or control empty vector (shV, Sigma-Aldrich, St. Louis, Mo.). Afterpuromycin selection, A549-shV and A549-shTLR5 cells were tested forNF-kB activation in response to CBLB502 treatment using luciferasereporter assay according to manufacture protocol (Promega, Cat#E4530,Madison, Wis.). Then A549-shV and A549-shTLR5 cells (1×106/100 μl ofPBS) were injected s.c. into 2 flanks of 20 athymic nude mice to inducetumors. Mice bearing s.c. growing A549-shV and A549-shTLR5 tumorxenografts (5 mice per group) were treated with either CBLB502 or PBSacting as control The results demonstrate that the repeatedadministration of CBLB502 alone led to a reduction in tumor growth ratesin the A549-shV (TLR5-expressing) tumor xenografts demonstrating adirect tumor suppressive effect of the drug. As shown for A549 derivedtumors, this effect was TLR5 dependent since TLR5 knockdown elicited bylentiviral transduction of shRNA against human TLR5 rendered the A549tumors no longer sensitive to the direct antitumor effect of CBLB502.Data are shown in FIG. 20.

Example 8

The effect of CBLB502 injections on H1299 tumors differing in TLR5expression (H1299-control vs. H1299-TLR5). In order to induce TLR5expression, H1299 cells (originally TLR5 negative) were transduced withlentriviral construct expressing human TLR5 gene. The functionalactivity of TLR5 was checked by IL-8 production in response to CBLB502treatment. Then both tumor cell types (1×10⁶/100 μl of PBS) wereinjected s.c. into 2 flanks of athymic nude mice to induce tumors.Similar to A549 model described above, mice bearing were treated witheither CBLB502 or PBS acting as control. The results demonstrate thatthe repeated administration of CBLB502 alone led to a reduction in tumorgrowth rates only in H1299-TLR5 (TLR5-expressing) tumor xenograftsdemonstrating a direct tumor suppressive effect of the drug. As shownfor the control H1299 (TLR5-negative) tumor growth was not affectedCBLB502 treatment. Data are shown in FIG. 21.

Example 9

This example demonstrates that bladder tissue is a strong responder toCBLB502. The experiment was conducted as described as described abovefor liver tissues. NF-kB dependent luciferase expression in liver, smallintestine (ileum part), colon, spleen, kidneys, lungs and heart wasassessed in the reporter mice 2 hs after s.c. injections of 100 μl ofeither PBS, CBLB502 (0.2 mg/kg) or LPS (1 mg/kg). Luciferase activitynormalized per μg of the protein extract was detected in 3 mice in eachgroup. The data are shown in FIG. 22.

Example 10

Table 2 shows the spectrum of genes transcriptionally activated byCBLB502 in target organs of mice (bladder results are shown). Genes thatare strongly upregulated in bladders of mice treated with CBLB502, 1 and3 hrs post-injection, are clustered according to their function. Thelargest group consists of chemokines, cytokines and their receptorsindicative of activation of innate immunity mobilizing mechanisms.

Example 11

CT-26 tumor cells, which do not express TLR5, were injected s.c. intosyngenic BALB/c mice to induce tumors. Tumor bearing mice were treatedwith CBLB502 (0.04 mg/kg, s.c.) given twice 24 hour apart. The volumesof s.c. growing tumors in treated mice were compared with tumors growingin the intact mice. Pre-treatment with CBLB502 did not have any effecton tumor growth. Then CT26 tumor growth was tested in the experimentalmodel of liver metastases induced by intrasplenic injection ofluciferase expressing CT-26 tumor cells (FIGS. 23B and C) and A20lymphoma cells (FIG. 23D) followed by splenectomy. Hepatic tumor growthwas assessed using Xenogen luciferase imaging every 4-6 days after thetreatment. Mice remained free from liver tumor growth were counted ateach imaging procedure. The results demonstrate prevention of tumorgrowth and significant delay of tumor appearance in livers by CBLB502treatment in both tumor models. The difference between CBLB502 treatedand control groups in liver tumor models (B, C, D) is significant (logrank p<0.05). The data are shown in FIG. 23.

Example 12

CBLB502 protection from Fas mediated hepatotoxicity. A. Survival ofNIH-Swiss mice after i.p. injection of 4 μg of anti-Fas antibodies aloneor in combination with CBLB502 (1 μg/mouse) injected 30 min, 2 hours and6 hours prior antibodies. In parenthesis are the numbers of mice pereach treatment. B. Protection of livers from anti-Fas antibody toxicity.Apoptosis in livers 5 hours after injections of anti-Fas antibodies wasdetected using TUNEL technique. Tissue morphology with H&E stainingrevealed necrotic damage to livers by anti-Fas antibody injections andprotection by CBLB502. Hemorrhage in liver was detected by erythrocyteinfiltration in tissue, mouse IgG control (purple) and DAPI nuclei(blue). Data are shown in FIG. 24.

Example 13

Liver protection from TNF-alpha and LPS toxicity. A. Caspases 3/7 weredetected 5 hours after injections of TNF-a or LPS and lipis oxidation(indicative of inflammation damage) was detected 24 hours post injectionin mice with and without CBLB502 treatment 30 min before TPS/TNF-a.Caspase activation and lipid oxidation in lungs induced by TNF (1mg/mouse) was prevented by CBLB502 injection. LPS (10 mg/kg) induceddamaging effect was completely abolished by CBLB502 injection 30 minbefore LPS. Data normalized by protein concentration, 24 hours after thetreatment, n=3. It was no caspase activation (5 hours after TNFinjections) and much less lipid oxidation (24 hours post-TNF injectionsas indicative of inflammatory damage) in livers of mice if CBLB502 wasinjected 30 min before h-TNF. B. Immunohistochemical analysis (H&Estaining) confirmed the preservation of liver integrity by CBLB502injection before TNF-a. Compared to the intact control, the liver of theTNF-treated mice showed vacuolization of the hepatocytes that isslightly more pronounced periportally and is dose-dependent (more severein TNF 0.4 mg/mouse). In the livers of mice treated with CBLB502 and TNF0.2 mg or 0.4 mg/mouse, the changes were minimal and the hepatocyteswere close to normal though slight vacuolization was still visible. Dataare shown in FIG. 25.

Example 14

Lung protection from TNF-a and LPS toxicity. Compared to intact control,the lungs of the TNF-treated mice showed reactive proliferation ofalveolar cells, hyperemia, interstitial edema and exudates in alveolileading to reduction of the air spaces and the alteration wasdose-dependent (more severe in TNF 400). In the lungs of mice treatedwith CBLB502 and TNF 200 ng or 400 ng, the changes were minimal. Themorphology was close to normal though slight thickening of alveolarwalls was still visible (FIG. 26B). It was almost normal level of lipidoxidation (indicative of inflammatory damage) in lungs of mice ifCBLB502 was injected 30 min before LPS (10 mg/kg) or h-TNF (0.05 mg/kg)(FIG. 26A). Data are shown in FIG. 26.

Example 15

Protection of mice from lethal oral Salmonella typhimuriumadministration by CBLB502 injections. Conditions of the experiments areshown in FIG. 27.

Example 16

This examples demonstrates that irinotecan abrogates the antitumoreffect of flagellin. The data are shown in FIG. 28. Fischer rats withs.c. growing syngeneic Ward colon tumors were treated with CBLB502 (0.2mg/kg), which was administered by i.p. once a day for three days.Irinotecan (200 mg/kg) was injected i.v. 30 min after each CBLB502injection. PBS was used as a vehicle control (FIG. 28A). CBLB502 rescuedrats from Irinotecan toxicity with no interference with irinotecanantitumor activity (FIG. 28B). The antitumor effect of CBLB502, however,was not observed in irinotecan-treated rats (FIG. 28C). Thisdemonstrates that the antitumor effect of CBLB502 requires sufficientinnate immunity levels.

TABLE 2 control flic 1 LPS 1 1 

3 

Chemokines Cytokines and their receptors CXCL2 1 4175.5 466.8 4175.5−1.6 92.4 113.7 92.4 CXCL10 1 3477.1 751.9 3477.1 6.1 304 18.9 49.8 CCL21 1460.8 623.6 1460.8 −0.6 247.6 −0.4 248.9 CXCL1 21.6 21314.9 13985.8986.8 73.3 18247.6 5458.7 248.9 CCL20 1 521.4 16.1 521.4 CXCL2 1 240.28.9 240.2 CCL7 25.2 3738.3 2575.3 148.3 1.1 664.1 218.3 10.7 CCL4 14.6613.2 3296.7 42.0 28.2 1002 3630.4 35.5 CCL3 1.8 74.2 350.5 41.2 CXCL911.6 185.6 94.5 16.0 2.8 389.7 481.3 139.2 CCL4 11.1 99.3 446.7 8.9 28.21002 3630.4 14.2 CXCL16 263.2 2048.1 546.6 7.8 36.8 524.2 386.1 14.2CXCL15 1 34.5 14.1 34.5 CCL5 19 480 658.7 25.3 CCL19 1.9 44.6 35.2 23.5CXCL12 −6.3 14.9 14.4 14.9 CCL25 26.5 224.5 280.1 8.5 CCL1 1 4.2 −2.54.2 CCL11 192.8 616.4 795.2 3.2 CCL11 149.7 462.1 565.6 3.1 CCL17 13.7126.5 114.3 9.2 3.5 540.1 373.7 154.3 CCL19 7.6 18.4 9.8 2.4 CCL2 11460.8 623.6 1460.8 CCL20 1 521.4 16.1 521.4 CCL22 1 3.9 7.7 3.9 CCL26 14.2 0.7 4.2 CCL28 1 3.7 −1.2 3.7 CCL3 1.8 74.2 350.5 41.2 CCL4 14.6613.2 3296.7 42.0 CCL4 11.1 99.3 446.7 8.9 CCL7 25.2 3738.3 2575.3 148.3CCL7 12.3 1453.5 1195.4 118.2 CCR3 1 2.3 2.8 2.3 CCR5 5.9 12.1 −6.2 2.1CCR5 11.5 22.7 11 2.0 CCR6 1 3.8 4.6 3.8 −4.7 17.5 5.8 17.5 CCRL2 62.7610.6 224.9 9.7 CLCF1 4.6 23.2 4.9 5.0 CNTFR 1 9.1 −1.1 9.1 CNTFR 14.529.8 21.8 2.1 CX3CL1 216.7 1081.9 328.9 5.0 183.2 1471.3 440.5 8 CXADR(CAR) 22.2 59.4 52.7 2.7 CXCL1 21.6 21314.9 13985.8 986.8 CXCL10 13477.1 751.9 3477.1 CXCL15 1 34.5 14.1 34.5 CXCL16 30.4 371.9 61.3 12.2CXCL16 263.2 2048.1 546.6 7.8 CXCL17 1 7.7 4.5 7.7 CXCL2 1 4175.5 466.84175.5 CXCL2 1 240.2 8.9 240.2 CXCL9 11.6 185.6 94.5 16.0 CXCR5 1.3 4.714 3.6 FAS 138.7 967.8 288.9 7.0 FAS 396.2 1802.5 730.6 4.5 FASL 3.610.2 1.1 2.8 FPR2 14.2 96.4 187.3 6.8 0.1 954.1 1765.8 954.1 IER3 1494.512012.4 8339.1 8.0 LIF 1 213.4 13.8 213.4 LIFR 1 6 4.7 6.0 DARC 131.2360.4 277 2.7 87.9 1102.6 2605.8 12.5 CMKLR1 −7.3 8.9 10.4 8.9 FPR2 0.1954.1 1765.8 954.1 EBI3 21.5 297.3 208.5 13.8 Interleukines and theirreceptors IL7R 1.6 24.9 43.8 15.6 IL8RA −2 10.7 3.4 10.7 IL9R 0.5 9.53.1 9.5 IL17C 1 887.3 31.8 887.3 LIF 1 213.4 13.8 213.4 IL1RN 1 52.845.2 52.8 51.3 729.5 538.5 14.2 IL1F5 −4.2 8 3.8 8.0 IL1R1 0.1 10 22.110.0 IL1R2 51.8 397.6 281.7 7.7 IL10 1 19.5 26.3 19.5 IL10RA 15.5 34.329.5 2.2 IL10RB 1.5 14 15.2 9.3 IL12A −1.2 32.2 10.3 32.2 IL12RB1 1 14.77.2 14.7 −0.5 13.7 10.9 13.7 IL12RB1 7.1 21.7 7.9 3.1 IL13 11.4 111.9258.5 9.8 IL13RA2 −4.2 12.4 121.7 12.4 IL15 4.8 10.6 8.6 2.2 IL15RA 7.438.1 19 5.1 IL15RA 5.1 24.8 14.3 4.9 IL15RA 19.2 47.1 32 2.5 IL16 1 2 42.0 IL17A 1 5.4 2.2 5.4 IL17B −2.5 30.8 −4.5 30.8 IL17C 1 887.3 31.8887.3 −0.2 8.6 1.7 8.6 IL17RA 26.1 72.2 37.1 2.8 IL17RB 0.2 38.1 10 38.1IL18BP 20 143.7 283 7.2 IL18R1 57.4 857.8 892.9 14.9 IL18RAP 1.3 24.30.1 18.7 IL18RAP 7.6 18.4 19.1 2.4 IL19 −0.7 26.7 95.4 26.7 IL1A 1 14.943.7 14.9 IL1A 2.8 35.5 72.3 12.7 IL1B 20.7 437.9 1023.3 21.2 IL1RAP 6.111.9 5 2.0 IL1RL1 1 22.3 13.1 22.3 IL1RN 1 52.8 45.2 52.8 −2.7 34.4 0.934.4 IL1RN 1.9 6 2.9 3.2 51.3 729.5 538.5 10.3 IL1RN 51.5 158.5 118.83.1 −2.9 10.3 −1 14.2 IL2RA −5.8 8.7 18.1 8.7 IL2RB 4.8 49 −2.2 10.2IL2RG 1.2 12.9 3 10.8 IL20RA 4.8 34.2 21 7.1 IL20RB 1.6 6 7.7 3.8 −1.111.9 −3.4 11.9 IL20RB 2.5 8.5 5.3 3.4 IL21R 1 7.5 3.3 7.5 5.8 65 65.411.2 IL21R 6.5 13 6.6 2.0 IL22RA1 1.2 8 3.1 6.7 IL23A 2.2 10.5 −2.1 4.8IL27 1 5.2 5 5.2 IL28RA 1.6 15.3 11.1 9.6 IL28RA 134.8 279.3 180 2.1IL2RG 1 7 6.1 7.0 IL31RA 1 4.5 7.6 4.5 IL33 150.4 352.9 222 2.3 IL4I128.9 1891 152.4 65.4 46.8 4000 741.7 85.5 IL5 1 2.5 0.4 2.5 −0.7 9.2 3.49.2 IL6 1.1 11.8 37.4 10.7 0.4 12 59.6 12.0 IL6RA 37.1 387.1 798.5 10.4ILF2 0.9 17.4 8 17.4 ILTIFB 0.8 20.5 10.4 20.5 Growth factors AIF1 −5.28.3 11 8.3 ARID5A 25.1 259.4 302.6 10.3 EGR4 1 231.2 65.7 231.2 CSF2 1202.5 17.1 202.5 0 10.4 4 10.4 CD70 1 54.7 9.8 54.7 0.6 520.2 67.5 520.2AREG 8 1299.1 256.3 162.4 11.5 692.1 255.8 60.2 CSF3-(GCSF) 1 35.4 15.635.4 CSF1 49.7 486 238.9 9.8 33.1 509.2 397.4 15.4 CSF2 1 202.5 17.1202.5 CSF2RB 1.2 6.6 −2.9 5.5 CSF3 1.3 6 12.8 4.6 CSF3-(GCSF) 1 35.415.6 35.4 CSF3R 1 2.3 0.3 2.3 EGR1 1824.5 6578.8 6395 3.6 EGR2 31.1224.1 157.4 7.2 EGR3 58.6 1126.3 1120.8 19.2 EGR4 1 231.2 65.7 231.2FGF10 3.5 8.8 8.3 2.5 FGF12 1 5.3 −5.8 5.3 FGF12 4.8 11.7 10.7 2.4 FGF134.9 15.2 6.9 3.1 FGF15 6.6 18.4 5.2 2.8 FGF17 1.1 3 2.1 2.7 FGF22 1.713.6 17.7 8.0 FGF3 1 2.3 9.7 2.3 FGFBP3 1 6.4 −2.5 6.4 FGFR1OP 11.8 31.716 2.7 FGFR2 11.1 24.6 19.5 2.2 GDF15 8.5 173.4 22.8 20.4 HBEGF 72.31118.3 246.3 15.5 IGFBP3 120.1 301.3 192.4 2.5 NELL2 −4.9 24.9 10.3 24.9BDNF 0.4 19.2 18.6 19.2 TGFBR1 6.6 73.4 137.8 11.1 NGFR 20.3 177.1 52.38.7 PAMP-recognizing molecules and other immune receptors PTX3 1 309.8525.7 309.8 −1.6 68.6 4.4 68.6 CLEC1A 5.8 12.5 9.5 2.2 CLEC2D 1547.24521 2234.1 2.9 CLEC2E 1 3.3 −1.4 3.3 CLEC4D 12.3 61.7 114.5 5.0 CLEC4E1 4.6 11.6 4.6 CLEC7A 5.4 11 13.2 2.0 CLECSF9 12.1 34.7 52.9 2.9 PGLYRP10 34.2 9.4 3.4 PGLYRP1 21.7 429.5 28 19.8 PTX3 1 309.8 525.7 309.8 PVR13.8 99.3 31.1 7.2 PVR 52.1 262.7 144.2 5.0 PVR 30.7 95.4 52.2 3.1 PVRL140.7 107.6 52.4 2.6 PVRL2 70.7 207.8 96.9 2.9 S100A8 34.5 288.9 32.6 8.4S100A9 23.6 139.9 20.6 5.9 SAA1 2.9 5.7 16.3 2.0 SAA3 125.6 500 1208.94.0 ICOSL 25.3 106.7 30 4.2 KLRG2 273.2 695.9 458.4 2.5 KLRI1 2.1 14.512.1 6.9 PRR, their adaptors and other receptor of innate immunity SFTPD−7.5 23.4 84.8 23.4 SAA3 69.9 3183.4 2571.7 45.5 PGLYRP1 40.8 2012.5 25549.3 KLRD1 5.8 107.8 171.1 18.6 HCST 26.4 198.3 202.3 7.5 FCGR4 37.61168.4 2200.4 31.1 MYD88 75.8 312.2 174.2 4.1 2.935748 6.730455 NOD2 5.337.6 17.2 7.1 2 30.3 7.8 15.15 0.408163 TLR1 5.1 TLR2 372.6 7545.62270.3 20.3 4.5 TLR3 9.1 TLR5 2.0 TLR6 2.804973 0.353293 TLR7 2.5 LY96332.8 2474.1 1466.9 7.4 IRAK3 109.4 314.9 231.3 2.9 101.8 941.3 1132.49.2 IRAK4 1 10.5 7 10.5 Anti-microbial proteins ZBP1 27.5 210.1 419.67.6 WFDC12 3.8 1134.8 3.3 298.6 S100A8 0 897.8 661.5 897.8 S100A9 −0.71021.2 1620.9 1021.2 RSAD2 154 5347.3 3471.3 34.7 REG3G −3.3 857.6 471.2857.6 MX2 21.9 959.8 671.8 43.8 LYZL4 24.8 215.1 −8.4 8.7 LTF −0.5 272184 272.0 DMBT1 −4.3 69.9 1 69.9 CRP 0.6 210.5 1721 210.5 CHI3L1 6.8843.8 257.1 124.1 HAMP 1 117.6 6.2 117.6 −7.2 27.2 8.9 27.2 LCN2 7.7492.4 173.8 63.9 9.2 1256.1 511.3 136.5 CHI3L1 11.8 128.2 46.5 10.9 6.8843.8 257.1 124.1 DEFB18 −5.7 11.7 −5.4 11.7 DEFB20 −0.5 22.1 22 22.1DEFB26 2.4 30.1 29.5 12.5 DEFB34 −2.2 7.1 −5.3 7.1 DEFB36 −0.7 8.3 −1.38.3 DEFB38 −6.9 7.6 −2.9 7.6 DEFB50 −5.4 8.6 6 8.6 DEFCR5 −2.6 10.9 −4.710.9 DEFCR-RS1 −1.2 8 3.9 8.0 DEFCR-RS12 −0.7 11.9 16.9 11.9 DEFCR-RS12−6 11.6 5.4 11.6 DEFCR-RS2 −2.8 9.4 −3.1 9.4 DEFB23 1 3.5 −1.9 3.5DEFB30 1 2.4 −1 2.4 DEFB5 7.4 16.4 12.4 2.2 DEFCR6 3.7 7.6 6.8 2.1 C727.4 57.7 20.1 2.1 CFB 24.2 396 682.4 16.4 C1QL2 −4.5 18.3 12.1 18.3 C91 4.2 2.2 4.2 −8 15.4 17.8 15.4 CAMP 1.2 7.5 12.4 6.3 IRGM1 1 3.6 3.43.6 1.2 32.2 −4.3 4.1 MX2 20 42.6 91.4 2.1 21.9 959.8 671.8 43.82648REG3G 1 81.2 3.9 81.2 −3.3 857.6 471.2 259.8 WFDC12 1 238.5 −2 238.5 3.81134.8 3.3 298.6316 LCN10 −0.7 49.8 50.7 49.8 LCN3 −1.3 36.5 0 36.5 CAMP2.4 56.3 43 23.5 APOM 1 21.7 2.5 21.7 APOOL −8 8.9 −8.2 8.9 C1QL2 −4.518.3 12.1 18.3 CD CD14 570.3 12181.7 2137.5 21.4 CD160 −1.1 70.5 96.270.5 CD177 1 5.7 −5.4 5.7 0.3 23.2 3.9 23.2 CD177 9.9 110.9 3.6 11.2CD19 2.1 6.9 5.7 3.3 CD200R1 1 4.1 −4.9 4.1 CD200R2 −4.5 7.6 12.1 7.6CD207 1.2 5.8 −2.4 4.8 CD207 1 3.1 2.4 3.1 CD247 1 2.9 3.6 2.9 CD27 −2.911.3 4.4 11.3 CD274 108 1070.2 398.3 9.9 CD28 1.2 2.7 1.1 2.3 CD209C 224.2 93.6 12.1 CD209D −3.6 35.5 54.7 35.5 CD209E −8.5 7.1 11.5 7.1CD300LB 1 2.7 −2.6 2.7 CD33 13.5 27.4 26.4 2.0 CD3E 17.2 122.3 21.2 7.1CD4 1.2 9.3 9.1 7.8 CD40 52.6 460.8 212.7 8.8 54.8 1419.4 775.7 25.9CD40 15.2 108.4 44.9 7.1 15.9 193.1 101.3 12.1 CD44 31.3 106.2 42.3 3.4−2.6 27.8 19 27.8 CD44 13.5 32.8 11.1 2.4 −1.7 22.8 0.2 22.8 CD44 305.7725.2 505.5 2.4 171.5 1251.9 596.8 7.3 CD44 66.4 133.3 77.7 2.0 CD52 12.1 −4.3 2.1 CD53 10 20.6 19.4 2.1 CD6 1 5.5 2.9 5.5 −2 7.4 17.5 7.4 CD61 3.3 −6.2 3.3 CD6 3.2 7.2 −1 2.3 CD6 6.7 13.1 13.5 2.0 CD69 4 100.3112.3 25.1 9.7 97.2 269.3 10.0 CD7 1.3 10.5 8.9 8.1 CD70 1 54.7 9.8 54.70.6 520.2 67.5 520.2 CD79B 1 3.9 4.6 3.9 CD80 8.5 31.5 16.2 3.7 CD80 3.17.1 3.5 2.3 CD83 45.7 771.8 486 16.9 CD86 3.2 5.8 −4.3 3.5 59.4 30.317.0 CD8B1 −1 30.2 −5.1 30.2 CD96 1 5.3 4.1 5.3 Transcription andproliferation NEO1 9.8 69.3 42.6 7.1 NFIC 12.3 136.2 297.6 11.1 POU2F211 161.3 164.9 14.7 POU3F1 6.5 140.6 83.1 21.6 SBNO2 54 765.1 860.1 14.2SIRT6 17.7 182.1 195.4 10.3 GTF3C6 4.2 84.2 106.1 20.0 GEMIN5 11.3 93.2157.1 8.2 FIP1L1 −2.1 57.9 10.3 57.9 FAP 5 51.7 2.4 10.3 CHD1 27.5 295.2529.8 10.7 BACH1 21.7 159 299.5 7.3 BARX2 15.6 234.6 144.6 15.0 ATF331.6 690.5 276.5 21.9 ATF4 43.8 115 71 2.6 BATF 17.2 150.8 57.3 8.8 21.9185.7 460.4 8.5 BAZ1A 27.6 93.8 44 3.4 CCND2 1.6 7.9 6.3 4.9 CCNL 30.367.6 38.5 2.2 CCNYL1 30.5 103.3 71.9 3.4 CDK2 29.1 67.9 52 2.3 CDK3 48.8 4.5 2.2 CDK5R1 24.2 110.2 42.6 4.6 CDK6 4.1 27.3 19.5 6.7 CDK7 1.53.8 −1.2 2.5 CDKN1A 147.1 374.4 226.9 2.5 CDKN1A 370.5 730.6 423 2.0CDKN2B 1231.3 2751.7 1575.8 2.2 EGLN3 406 2015 751.8 5.0 −1.3 19.3 −4.619.3 EGLN3 3.2 11.4 1.1 3.6 EIF4E1B −2.4 18.9 4.9 18.9 ELF3 #REF! 3525.2875.2 14.4 ETS1 1 22.7 7.7 22.7 FKHL18 63.6 336.2 133.8 5.3 52.6 431.6355 8.2 FOSB 92.3 738.1 332.9 8.0 FOSL2 4.6 32.2 8.5 7.0 0.4 11.7 −0.111.7 FOSL2 4.7 20.4 6.8 4.3 6.7 70.5 61 10.5 FOXA3 1 13.2 6.6 13.2 3.2100.6 0.9 31.4 FOXB1 1 3.8 9.2 3.8 FOXD2 1 5.6 4.7 5.6 1.2 10.3 40.5 8.6FOXE1 −0.4 8.8 −2.9 8.8 FOXF2 −1.2 10.3 20.6 10.3 FOXF1A 400.7 960.3831.7 2.4 FOXI2 1 2.6 −3.8 2.6 FOXJ3 9.2 20.3 13.9 2.2 FOXJ3 5.7 12.2 92.1 FOXK1 2.9 7.7 5.2 2.7 FOXK1 2.6 5.4 −1.3 2.1 FOXL2 1 9.5 0.1 9.5−1.6 13.3 4.4 13.3 FOXO4 −6.2 10.5 12.5 10.5 FOXO6 1 2.8 0.9 2.8 0.330.4 99.7 30.4 FOXP3 0.9 19.6 3.9 14.1 FOXP4 21 69.7 18.7 3.3 G3BP1350.1 898.7 467.6 2.6 CXXC1 9.4 25.7 18.5 2.7 GADD45A 1324.4 6076.12042.7 4.6 GADD45A 903.7 3390.1 1019.9 3.8 GADD45B 1 72.8 70.2 72.8 1.8101.6 60.8 56.4 GADD45B 106.8 2799 1357.3 26.2 GADD45G 498 1323.4 1534.72.7 HIF1A 245.5 704.1 335.7 2.9 HIF1AN −3.2 23.9 41 23.9 JDP2 140.5635.1 586.8 4.5 JUN 128.1 714 553.2 5.6 JUNB 994.9 2976.3 3927.9 3.0JUND1 72.6 227 113.1 3.1 MAFF 2.5 72.5 17.3 29.0 NF1 1 14.4 2.8 14.4NFATC1 6.6 58 11.3 8.8 NFATC1 15.8 75.5 35.2 4.8 NFATC1 152.8 550 312.63.6 NFATC1 179 586.1 275.9 3.3 NFATC2 1 8.4 9.3 8.4 NFATC2 1 5.9 −2.35.9 NFE2L2 403.6 1300.1 834.9 3.2 KLF6 32.4 74.9 74.9 2.3 MYC 48.9 160.6153.1 3.3 MYCT1 1 4.8 0.7 4.8 SOX7 17 43.5 44.9 2.6 SOX9 1 140.4 23.5140.4 SOX9 109.4 2159.4 462.5 19.7 Apoptosis ANKHD1 35.7 320.8 506.2 9.0AEN 15.1 41.5 16.7 2.7 APBA1 0.3 14.8 7.9 14.8 APBB1IP −3.9 11.3 80 11.3APH1A 0.1 140.8 158.1 140.8 APIP 15.4 119.7 190 7.8 BIRC2 25.6 222.3277.1 8.7 BIRC3 −4.7 47.2 2.2 47.2 BIRC6 −0.9 9.4 −1.7 9.4 BBC3 −1.159.9 131.2 59.9 BCLAF1 −1.6 36.8 2 36.8 BCL10 1309.4 3434.1 1987.1 2.6BCL10 2.9 6.6 8.9 2.3 BCL2A1B 60.8 330.2 280 5.4 BCL2A1C 3.3 27.3 19.28.3 BCL2A1D 1 15.8 7.6 15.8 BCL2A1D 32.6 253.6 247.7 7.8 BCL2L11 5 37.35.6 7.5 BCL2L11 50.7 302.2 52.2 6.0 BCL2L11 22.7 115.2 27.8 5.1 BCL2L11455 1485 306.3 3.3 BCL3 32.9 610.6 270.4 18.6 BCL6B 47 97.6 112.8 2.1BCL9 3.6 13.6 16.5 3.8 BCL9L 1.3 21 12.7 16.2 BCOR 209.5 980.2 226.5 4.7CASP4 125.3 740.5 278.1 5.9 CASP8 404.5 845.1 530.3 2.1 NUPR1 1049.63339.3 2496 3.2 DNASE1L3 8.9 179.6 139.6 20.2 PDCD4 476.3 4862.7 12537.810.2 Kinases CSNK1G1 1 8 18.3 8.0 0.5 75.7 119.2 75.7 DUSP1 1719.34023.2 4344.7 2.3 DUSP16 48.9 143.8 66.2 2.9 DUSP13 −3 23.8 27.7 23.8DUSP2 30.1 262.3 191.1 8.7 8.2 78.6 58 9.6 DUSP2 21.8 106.3 81.9 4.920.3 180.1 51.3 8.9 DUSP3 49.6 101.7 105.6 2.1 DUSP3 40.1 81.1 125.2 2.0DUSP6 20.4 161.5 35.7 7.9 DUSP6 50.9 174.7 46.3 3.4 DUSP6 1275.3 3870.61335.9 3.0 DUSP8 96.2 454.9 414.5 4.7 MAP2K3 1864.1 4967 2371.6 2.7MAP3K2 2.5 6.8 7.2 2.7 MAP3K7 34.9 74.5 51 2.1 MAP3K8 52.5 618.6 432.511.8 MAP4K5 2.6 5.8 2.4 2.2 0.5 8 8 8.0 MAPK11 122.6 248.5 139.9 2.0MAPK15 1 3.6 0.6 3.6 MAPK1IP1L 183.6 365 286.5 2.0 MAPK6 477.4 986.2578.6 2.1 MAPK8 4.9 11 6.9 2.2 MAPK8 1 2 0.5 2.0 MAPK8IP3 1 14.7 7.814.7 MAPK8IP3 2 5.6 7 2.8 MAP2K1 15.1 120.9 127.1 8.0 MAP3K12 7.7 58.766.7 7.6 MAP3K4 0.2 8.9 17.1 8.9 MAP3K5 −2.5 19.4 12.2 19.4 MAP3K6 59.4475.2 741.5 8.0 MAPK10 −1 12.9 40.2 12.9 MAPK7 0.7 61.3 1.4 61.3MAPK8IP2 1.1 38 0.6 34.5 CKMT1 −1 61.6 144.9 61.6 CSNK1G1 0.5 75.7 119.275.7 DAPP1 21.1 236.3 160.8 11.2 DYRK1A 4.9 80.1 87.6 16.3 PIM1 92.9227.5 130.4 16.7 PIM1 434.4 1213.4 801.2 3.2 PIM2 92.9 227.5 130.4 7.3PIM2 434.4 1213.4 801.2 2.1 PLK2 92.9 227.5 130.4 2.4 PLK3 434.4 1213.4801.2 2.8 ITPKC 405.2 1250.1 984.1 3.1 RIPK2 86.5 856.8 300.4 9.9 79.7819.8 152.4 10.3 Cathepsins CTSB 2.8 6.9 4.8 2.5 CTSC 9.2 64 7.1 7.0 40410.5 164.3 10.3 CTSC 7.9 37 5.3 4.7 7.8 60 0.3 7.7 CTSC 39.3 134.5 42.73.4 17.9 136.5 34.8 7.6 CTSJ 1.1 2.4 −3.6 2.2 0.9 11.9 3.9 11.9 LAMP2 221.3 14.4 10.7 CTSM −3.8 11.1 73.6 11.1 CTSR 10 78.6 88.2 7.9 Interferonand IFN-inducible genes IRF1 16.4 531.5 170.4 30.7 GBP1 796 1974.11706.5 2.5 GBP10 8.6 53.9 40.6 6.3 GBP2 28.9 67.5 76.4 2.3 GBP3 580.41978.1 1291.3 3.4 GBP3 548.9 1582.2 1065.5 2.9 GBP5 90.4 672 507.1 7.4IFI202B 14.6 51.4 60.1 3.5 IFI47 38.6 90.7 95.3 2.3 IFITM1 3562.3 8026.15161.6 2.3 8.1 474 585.6 58.5 IFITM1 1023 7728.1 4581.7 7.6 IFITM5 1.320.3 10.7 15.6 −2.5 14.6 −1.9 14.6 IFITM6 −1.9 12.1 62.7 12.1 IFNE1 8.528 3.1 3.3 IFNG 1 2.6 3.8 2.6 IFNGR2 107.1 425.9 202.8 4.0 IRF5 23.552.3 54.6 3.9 IRF6 23.5 52.3 54.6 4.1 −5.1 15.4 −4.7 15.4 IRF6 23.5 52.354.6 2.0 IRF7 15.4 230.7 207.4 15.0 IRF9 23.5 52.3 54.6 2.2 PLSCR1 114.6893.3 318.7 7.8 PLSCR1 66.9 305.6 158.3 4.6 SLFN2 18.4 252.7 69.5 13.7ISG20L1 19.9 159.7 166.4 8.0 IGTP 164.5 1836.5 1147 11.2 LOC100048583−3.5 27.8 109.6 27.8 LOC100048583 −3.5 27.8 109.6 20.4 GVIN1 45.4 458.2976.1 10.1 proteasome PSMB9 15.4 114.6 142.3 7.4 PSMD11 6.1 62.8 0.610.3 PSMD3 −5.7 15.5 132.5 15.5 Ubiquitine-associated CUL4A 1 3 −0.2 3.0IBRDC3 177 699 463.2 3.9 LNX1 1.4 10.9 6.2 7.8 UBD 17.4 85.8 20.6 4.9UBTD2 20.4 60.9 52.8 3.0 USP2 6.2 15.6 20.9 2.5 USP23 2.7 8.7 8.2 3.21.4 28.6 −1.8 20.4 USP25 6.9 15.6 18.6 2.3 USP37 5.5 19.2 7.9 3.5 USP3822.9 45.5 27.7 2.0 USP42 1 2.9 5.2 2.9 USP48 4.8 17.1 13.8 3.6 USP27X−0.9 8.7 −1.5 8.7 USP29 −2.8 7 10.1 7.0 USP37 19 330.9 294.3 17.4 USP4310.1 108.8 30.8 10.8 USP8 −2.4 27.9 5.3 27.9 USP9X −3 37.8 115.3 37.8CUL4A 0.5 22.4 6.2 22.4 UBD 13.4 446.1 278.5 33.3 LINCR 24.7 1194.7109.8 48.4 DTX3L 166.3 1484.1 608.4 8.9 MARCH1 10.5 75.1 134.7 7.2Actin-tubulin FLNB CCT5 288.6 2407.1 1216.1 8.3 CDC42EP1 13.6 110.1113.6 8.1 CDC42EP5 2 52.5 48.1 26.3 CEP350 −2.3 54.4 167.6 54.4 ARPC419.9 155 138.7 7.8 ARPM1 −1.1 10 3.2 10.0 ACTB 3142.5 7089.1 6166.2 2.3ARC 16.7 320.9 83.6 19.2 ABLIM3 1 4.8 7.9 #REF! −5.9 17.4 13 17.4 ACTL7B−2.4 13.6 0.9 13.6 ACTR6 −1.4 9.4 28.3 9.4 SCIN 54 597.9 49.6 11.1 59.71853 145.9 31.0 TUBA6 1429.6 2863.9 2070.6 2.0 TUBB2B 940.9 2963.81731.2 3.1 ER-transport DUOXA2 1 280.8 5.7 280.8 11.5 878 25.2 76.3 EHD1131.3 725.6 270 5.5 115.1 808.1 615.6 7.0 G-proteins etc GPR109A 131.74123.4 1185.8 31.3 GPRC5A 39.6 421.2 251.8 10.6 RAI3 24.4 212.5 117.68.7 LPAR2 32.4 221.4 19.3 6.8 LPAR3 129.6 660.7 158.6 5.1 RGS16 RGS16387.9 372.6 13.4 RND1 1.6 11 20.7 6.9 RND3 157 806.7 435.3 5.1 GPR84 165.4 70 65.4 GNAS −1 267.2 553.9 267.2 LPAR2 12.3 94.5 −3.7 7.7 Mucosegeneration and cell-to-cell connection KRT16 1 150 8.2 150.0 HAS1 22.7939.7 1378.5 41.4 SPRR2G 1.8 70 11.3 38.9 9.6 817.1 19.2 85.1 PCDH10 138.4 7.4 38.4 −0.9 89 90.9 89.0 PCDH10 −1.4 9.9 11.2 9.9 PCDHA11 1.442.8 14.2 30.6 PCDHA7 −1 10.6 −3.3 10.6 PCDHA7 6.1 50 98 8.2 PCDHB6 214.1 29.5 7.1 PCDHGA10 1.8 17.8 55.1 9.9 PCDHGA9 −0.5 11.8 0.7 11.8PCDHGB6 −4 7 −8.3 7.0 PCDHGB7 −2.9 8.3 62.3 8.3 PCDHGB8 −3.1 16.3 7.816.3 AMIGO2 205.4 480.7 564.1 2.3 CATNAL1 1.9 14.3 4.2 7.5 CHST4 1 3.82.1 3.8 CLDN7 91.3 187.6 110.1 2.1 104.5 794.3 479.9 7.6 CNFN 1 19.3−10.6 19.3 −1.5 74.4 −4.8 74.4 COL11A1 1 4.7 1.5 4.7 0.5 15.2 59.2 15.2COL11A1 1 2.6 0.3 2.6 COL11A2 9.5 19.9 15.5 2.1 COL19A1 1 4.5 −0.6 4.52.1 43.4 5.9 20.7 COL25A1 6.6 12.9 13.7 2.0 COL2A1 2.3 5.8 11 2.5 COL4A32.9 9.9 7.1 3.4 COL5A3 6.6 21.6 11.4 3.3 COL6A1 11.1 27.4 19.1 2.5COL9A1 1.9 3.9 −3.1 2.1 COL9A2 2.3 12.2 9.8 5.3 COL5A2 2.8 70 90.4 25.0COLQ −6.9 14.5 7 14.5 FAT1 6.9 28.2 31.2 4.1 FLNB 329.5 729.2 391 2.2301.2 2663.6 1356 3.3 GALNT3 1 26.3 −1.5 26.3 GFPT1 1 14.5 6.6 14.5 0.916.6 8.4 16.6 GFPT1 3.6 14.4 5.6 4.0 14.6 105.7 33.8 7.2 GFPT2 155.11284.9 1257.2 8.3 97 1762.3 2584.6 18.2 GCA 9.4 24.9 20.6 2.6 GJA10 27.3 12.3 3.7 GJA8 1 4.9 2.6 4.9 GJB1 7.3 14.8 13.7 2.0 GJB2 26.5 58.638.3 2.2 GJB2 364.8 770.8 479.5 2.1 GJB2 33.3 68 46.7 2.0 GJB4 1 16.56.2 16.5 GJC2 1 3.5 3.6 3.5 1.1 13 45.5 11.8 GJD2 1 4.6 −1 4.6 −5 18.351.1 18.3 HAS1 22.7 939.7 1378.5 41.4 HAS2 11 81 88.7 7.4 HS3ST1 753.48916.3 1840.7 11.8 HS6ST1 5.3 25.1 −0.2 4.7 HS6ST1 395.4 1824.5 349.24.6 ITGA2 5.6 49.3 13.1 8.8 ITGA5 157.4 332.9 164.5 2.1 ITGA6 1 9 4.19.0 ITGAD 1.5 3 −0.3 2.0 2.1 18.2 −5.4 8.7 ITGAE 1 10 8.4 10.0 ITGAV59.2 172.4 86.5 2.9 ITGB2 2.7 53.1 8 19.7 ITGB6 180 620.7 249.4 3.4ITGB6 1253.2 3474.8 1568.1 2.8 ITGB6 513.8 1370.7 645.1 2.7 ITGP 1 10.64.8 10.6 KRT14 254.8 1063 660.2 4.2 KRT1-5 1 18.6 −2.1 18.6 KRT16 1 1508.2 150.0 1.2 148 16 123.3 KRT23 679.4 4234.8 720.2 6.2 KRT36 6.5 60.52.5 9.3 KRT17 7.7 103.2 65.5 13.4 KRT33B −1.8 7.9 0.8 7.9 KRT35 −4.412.1 85.9 12.1 KRT82 −0.6 18.2 3.6 18.2 KRT84 0.8 8.1 20.4 8.1 KRT86−1.7 12.9 4.6 12.9 KRTAP16-5 0.3 14.1 34.6 14.1 KRTAP3-2 −2.7 7.9 −5.97.9 KRTAP9-1 −1 7.7 49 7.7 KRTDAP 19.2 157.7 258.3 8.2 LOXL4 2.7 21.67.6 8.0 SPRR2D 6.1 1052.3 −0.1 172.5 1.1 3215.9 14.1 2923.5 SPRR2E 1245.5 −8.1 245.5 −1.9 772.1 −4.5 772.1 SPRR2F 14.4 137.8 9.1 9.6 5.92404.5 45.6 407.5 SPRR2G 1.8 70 11.3 38.9 9.6 817.1 19.2 85.1 CDCP1 5.843.7 21.8 7.5 AMICA1 22.2 372.2 455 16.8 CDH2 10.2 74.1 10.8 7.3 LOXL443 469.9 373.6 10.9 NRXN2 6.5 78.9 153 12.1 Immune adhesion moleculesVCAM1 5.4 68.1 34.9 12.6 0.8 45 −6.6 45.0 VCAM1 637.4 4462.9 3422.1 7.00.9 14.9 9.4 14.9 ICAM1 329.6 7607.4 4220.4 23.1 256.4 2069.1 705.5 8.1SELP 36.5 225.9 1068.5 6.2 28 756.9 2978.1 27.0 SELL 4.3 10 6.6 2.3 8.884.1 26.8 9.6 CEACAM1 12 137.2 72.8 11.4 CEACAM1 42.4 303.9 59.4 7.2CEACAM2 50.4 408.1 22 8.1 NF-kB and inflammation REL 1 132.3 26.3 132.3NFKBIZ 147.6 6159.8 2878.9 41.7 NFKBID 40.9 1493.2 362.5 36.5 IKBKE 90.1569 99.7 6.3 NFIB 3.5 9 5.6 2.6 NFKB1 20.5 151.3 76.4 7.4 7.4 69.9 1449.4 NFKB1 561.3 2247 840.7 4.0 NFKB2 1 6.8 −0.5 6.8 NFKBIA 630 11386.86795.9 18.1 18.1 301.2 226.9 16.6 NFKBIA 27.7 338.8 182.1 12.2 NFKBIB19.8 200.7 46.8 10.1 17.9 264.1 221.6 14.8 NFKBID 40.9 1493.2 362.5 36.527.8 399 248.8 14.4 NFKBIE 8.1 180.2 29.6 22.2 60.2 536 169.6 NFKBIE60.2 731.7 136 12.2 164.2 3145.7 2250.7 3.9 NFKBIZ 147.6 6159.8 2878.941.7 19.2 REL 1 132.3 26.3 132.3 RELA 1948.7 4768.8 2897.9 2.4 RELB106.2 1265.6 336 11.9 HSP HSPA1A 64.7 744.8 102.8 11.5 HSPA1A 35.2 239.853.7 6.8 HSPA1A 1469.1 5421.2 1074.3 3.7 HSPA1B 22.6 227.3 22.7 10.1HSPA1B 135 1184.7 193.7 8.8 HSP90AA1 6.6 49.3 77.1 7.5 HSPA14 −11.8 8−3.9 8.0 HSPB3 −0.4 19.8 44.3 19.8 COX4I2 22.2 236.9 358.2 10.7 LTB4R128.9 222 363.3 7.7 NKIRAS1 14.9 135.5 189.4 9.1 RIPK2 79.7 819.8 152.410.3 Ion-channel KCNE2 1.8 68.8 4.5 38.2 MCOLN2 12.8 42.3 26.9 3.3 7.2108.2 7.9 15.0 CYCS 33.1 66.2 43.6 2.0 10.2 116.5 193.5 11.4 CLIC4 121.4459 228.6 3.8 CLIC6 33.9 494.6 73.1 14.6 CACNA1F 1 10 4.1 10.0 CACNG3−8.9 9.4 −0.8 9.4 CACNG5 −1.6 16.1 47.9 16.1 CACNG6 1 13 7.9 13.0 CLCA35.3 46.1 −4.1 8.7 CLCN3 1.3 10.7 −2.6 8.2 CLCNKA −0.8 7.3 5.3 7.3 CLIC633.9 494.6 73.1 14.6 KCNA10 2.3 20 6.5 8.7 KCNA7 0 14.2 −3.8 14.2 KCNAB1−3.3 8.4 −4.2 8.4 KCNC2 −2.5 20.1 −0.5 20.1 KCND2 2.8 26.3 3.4 9.4 KCND3−6.3 9 −0.1 9.0 KCNE1L 6.8 70.1 112.6 10.3 KCNH4 −0.8 9.4 13.3 9.4 KCNJ1−3 8 6.4 8.0 KCNK13 23.7 216.8 298.4 9.1 KCNK15 0.5 7.7 40.7 7.7 KCNK9−4.4 9.8 2.4 9.8 KCNQ2 −1.3 9.1 10.3 9.1 KCNQ2 −1.3 9.1 10.3 7.7 KCNQ5−2.1 20.3 −1.2 20.3 KCNS3 −1 11.6 4.7 11.6 SCN2A1 1 14.8 −7 14.8 SCN8A−8.8 13.5 16.4 13.5 SCNM1 9.4 172.1 333.6 18.3 SCNM1 26.5 214.3 284.78.1 TPCN1 −5.1 12.8 4.5 12.8 MCOLN2 7.2 108.2 7.9 Histone and histoneassociated EHMT2 1 10.1 14 10.1 DOT1L 16.5 56.1 42.6 3.4histocompatibility 2 H2-Q5 10.7 171.7 59.7 16.0 H2-Q7 124.9 313.3 141.12.5 H2-Q7 30.4 73.3 42.7 2.4 H2-Q8 17.2 96.9 34.2 5.6 Ras-Rho relatedRAN 45.6 RANBP3L 13.8 RANGAP1 14.0 CDC42EP2 732.4 2299.1 556.7 3.1 CDGAP1.9 7.7 7 4.1 IRGQ 562.2 1304.5 491.8 2.3 RAB20 4.9 27.8 29.5 5.7 RAB32452.5 1946 939.8 4.3 RHOF 17.9 252.9 14 14.1 21 340.1 5.1 16.2 RAB1 8.7RAB1B 15.4 RAB3B 9.5 RAB3IP2-PENDING 20.9 RAB5B 13.2 RAB7 12.4 RAP1A11.7 RASAL1 7.0 RASGEF1A 14.6 RASGRP1 14.9 RASSF10 15.3 RASSF4 9.7RASSF6 10.5 RASSF9 12.9 ARHGAP15 7.2 ARHGAP25 9.8 ARHGAP5 9.0 ARHGEF17.9 ARHGEF7 11.2 Protease-inhibitor SERPINA3F 1 19.9 −0.3 19.9 96.3SERPINA3G 142.5 5320.8 1240.8 37.3 SERPINA3H 31.7 2296.8 799.4 72.5287.3 SERPINA3N 675 1797.8 1726.1 2.7 30.0 SERPINB1A 45.3 88.7 121.7 2.0SERPINB2 1 79.1 358.6 79.1 SERPINA12 11.3 SERPINA1C 11.0 SERPINA3G 129.7SERPINA3M 20.3 SERPINB3B 12.3 SERPINE2 11.1 SERPINF2 13.2 CST6 −4.4107.1 74.5 107.1 STFA1 4.9 67.2 22.5 13.7 solute carrier family SLC10A50.2 27.5 1.7 27.5 SLC10A6 78 434.6 617.3 5.6 SLC10A6 30.9 155.7 163 5.077.3 592.2 321.4 7.7 SLC11A2 6.1 32.2 20.7 5.3 52.9 503.5 229.3 9.5SLC11A2 60.3 147.6 106 2.4 SLC12A1 5 70.3 94.4 14.1 SLC12A2 −6.7 7.4−0.3 7.4 SLC13A3 1.3 10.1 3 7.8 SLC14A2 −4.4 29 23.9 29.0 SLC15A3 27.3135.7 73.2 5.0 23.6 260.3 476.5 11.0 SLC16A3 5.1 14.5 7.3 2.8 SLC16A554.2 183.9 66.3 3.4 45.2 417.1 80.3 9.2 SLC16A9 175 473.1 267.9 2.7 1.2172.9 315.5 144.1 SLC17A3 6 62.8 9.2 10.5 SLC17A6 −2.9 8 −5.8 8.0 SLC1A4194.9 460.9 191.4 2.4 SLC25A25 195.8 956.8 660.4 4.9 SLC2A6 62.8 3525.2871.1 45.1 SLC22A3 −1.1 19.2 55.1 19.2 SLC22A4 2.4 52.3 −4.8 21.8SLC22A6 −0.8 9.9 −2.3 9.9 SLC22A9 −8.3 24.4 −6.1 24.4 SLC24A5 −1 45.3 1645.3 SLC25A2 0.7 10 4.6 10.0 SLC25A34 −4.9 7.5 −0.9 7.5 SLC25A4 0.9 8.1−2.2 8.1 SLC25A40 −1 17.1 3.2 17.1 SLC25A40 −2.6 10.4 16.6 10.4 SLC26A3−4.5 8.8 8.1 8.8 SLC26A4 3.1 45.5 10.6 14.7 SLC26A7 −9.7 15.9 5.1 15.9SLC29A4 4.6 41.7 23.7 9.1 SLC2A2 −6.5 12.7 8.2 12.7 SLC2A6 62.8 3525.2871.1 56.1 SLC30A3 2.3 52.9 2.1 23.0 SLC30A4 −0.3 34.6 57.5 34.6 SLC34A20.1 7.3 −3.2 7.3 SLC35D3 −4.1 12.3 −4.5 12.3 SLC35F4 −0.6 9.9 63.5 9.9SLC36A3 0 34.6 5.4 34.6 SLC38A1 −0.5 18.3 13.7 18.3 SLC38A2 3.8 35 8.19.2 SLC39A8 29.3 77.6 105.2 2.6 SLC39A8 −10.1 10.6 −2.6 10.6 SLC45A3 31169 46 5.5 SLC39A4 129.2 927.6 400.6 7.2 SLC39A5 −2 31.7 2.4 31.7 SLC3A2−0.2 26.2 0.5 26.2 SLC45A2 0.3 16.2 18.4 16.2 SLC4A1 21.3 212.3 266.810.0 SLC4A4 5.7 171.3 291.7 30.1 SLC5A1 37.8 307 107.7 8.1 SLC5A10 −228.6 −0.6 28.6 SLC5A2 −2.3 17.3 −1.1 17.3 SLC6A12 −0.3 8.7 64.1 8.7SLC6A15 −6.3 40.3 5.6 40.3 SLC6A16 −6.5 8.2 3 8.2 SLC6A2 0.2 14.9 6.814.9 SLC6A4 −3.3 10.7 98.3 10.7 SLC7A11 2.7 29.1 13.1 10.8 SLC7A11 44.5408.4 38.8 9.2 SLC7A2 −4.2 8.1 2.7 8.1 SLC7A9 −3 15.3 13 15.3 SLC8A2 0.88.6 15.2 8.6 SLC9A6 2.2 26.1 47.3 11.9 SLCO1A5 1.4 15.1 6.1 10.8 SLCO4A1−2.7 180.6 39.5 180.6 SLCO6B1 −6.4 7.6 51.4 7.6 Suppressor of cytokinesignaling SOCS1 4.7 56.2 61.4 12.0 SOCS2 209 1043.8 941.3 5.0 SOCS3177.3 4410.8 5007.3 24.9 52.5 2767.6 3346.5 52.7 SOCS4 69.3 211.4 94.83.1 SOCS7 −3.2 7.9 4.1 7.9 SOCS7 −1.6 7.3 −6.7 7.3 olfactory receptorOLFR1 −3.2 7.1 1.6 7.1 OLFR100 2.7 19.9 8.7 7.4 OLFR1000 −5.2 8.3 7.58.3 OLFR101 −5.4 10.2 2.4 10.2 OLFR1015 0.3 37.5 0.5 37.5 OLFR1024 −420.4 −5 20.4 OLFR1038 −3.4 7.6 7.6 7.6 OLFR1040 1.7 13.9 15.2 8.2OLFR1042 −1.1 19.6 8 19.6 OLFR1048 −1.1 12.5 −1.6 12.5 OLFR1056 0.7 164.2 16.0 OLFR1061 −1.9 11.1 8.5 11.1 OLFR1065 −1.2 15.3 17.3 15.3OLFR1085 −2.4 27.6 107.2 27.6 OLFR1102 1 12.7 0.5 12.7 OLFR1109 −4.116.4 7.3 16.4 OLFR1112 −5.9 20.7 3.3 20.7 OLFR1129 −7.3 7.3 −1.9 7.3OLFR113 −5 31.6 37.9 31.6 OLFR1130 −1.1 16 4 16.0 OLFR1133 1.3 9.9 11.37.6 OLFR1138 −7.9 11.7 16.8 11.7 OLFR1148 −3.8 7.8 1.4 7.8 OLFR115 −111.3 14.6 11.3 OLFR1170 −2 10.6 63.8 10.6 OLFR1176 −3 8.9 4.5 8.9OLFR1178 1.1 9.9 −4.8 9.0 OLFR1181 0.3 7.5 57.7 7.5 OLFR1183 −6 18 3.718.0 OLFR1186 −4.3 8 −2.6 8.0 OLFR1198 3 52.2 70.4 17.4 OLFR120 −5.715.5 96.7 15.5 OLFR1223 0.8 11.4 −10.5 11.4 OLFR1232 1.5 11.5 −1.3 7.7OLFR1249 3.9 50.6 −1 13.0 OLFR1250 1.8 23.6 56.1 13.1 OLFR1260 −4.2 10.79.6 10.7 OLFR1278 −1.2 10.2 6.6 10.2 OLFR1288 −2.2 8.1 10.9 8.1 OLFR1309−2.1 7.6 0.9 7.6 OLFR1320 −2.9 8.4 0.1 8.4 OLFR1323 −4.5 12.1 0.6 12.1OLFR1331 1.3 11 31.5 8.5 OLFR1333 0.1 41.1 92 41.1 OLFR1337 −2.8 26.1−5.2 26.1 OLFR1342 −4.2 10.8 49.8 10.8 OLFR1344 −7.4 9.4 27.9 9.4OLFR1347 −10.5 8.1 5.3 8.1 OLFR1348 −3.5 10.7 3 10.7 OLFR1349 −0.5 13.24.7 13.2 OLFR1361 1.2 9.8 −0.3 8.2 OLFR1384 −1.8 45.5 19.6 45.5 OLFR1385−6.9 12.3 11.6 12.3 OLFR1390 −0.4 24.7 82.9 24.7 OLFR1406 −3.1 7.1 −5.57.1 OLFR1412 3.3 49.4 4.3 15.0 OLFR1424 1.7 44.3 66 26.1 OLFR1436 0.916.6 40 16.6 OLFR1437 2.8 41.2 26.6 14.7 OLFR1443 −1 15.7 49.7 15.7OLFR1444 −2.3 34.1 31.4 34.1 OLFR1453 1.4 13.2 38.9 9.4 OLFR1474 0.315.9 −4.6 15.9 OLFR1475 0.1 15.9 39.3 15.9 OLFR148 −1.6 7.7 −2.9 7.7OLFR1489 −1.5 22.2 16 22.2 OLFR1508 1 8.7 11.5 8.7 OLFR1513 −4.3 14.711.8 14.7 OLFR159 3.6 91.1 18.3 25.3 OLFR165 −1.9 14.4 −3.6 14.4 OLFR166−0.5 9.2 2.5 9.2 OLFR167 −4.1 20.6 −1.7 20.6 OLFR168 −3.9 36.2 54.1 36.2OLFR168 0.3 9 21.7 9.0 OLFR171 −3.2 14.2 5 14.2 OLFR176 −3 22.6 15.222.6 OLFR192 −3.6 20.7 60.7 20.7 OLFR257 −1.6 31.9 22 31.9 OLFR26 1 13.213.7 13.2 OLFR262 −1.9 8.3 4.8 8.3 OLFR270 3.3 25.7 93.6 7.8 OLFR272 4.549.8 110 11.1 OLFR293 −0.1 21.2 −3.4 21.2 OLFR31 0.2 14.9 24.6 14.9OLFR312 −2.6 10.4 −6.3 10.4 OLFR313 0.7 15.7 58.4 15.7 OLFR351 −6.3 2466.5 24.0 OLFR362 −8.3 20.3 30.4 20.3 OLFR376 0.7 8.3 −4.9 8.3 OLFR380−3.8 9.1 −0.7 9.1 OLFR415 −0.3 20.3 17.7 20.3 OLFR429 −2.4 14.3 −0.414.3 OLFR432 −10.6 7.3 14.8 7.3 OLFR434 3 48.6 16.7 16.2 OLFR458 −2.521.4 36.6 21.4 OLFR464 −0.7 14.4 73 14.4 OLFR467 −2.2 15.1 16.4 15.1OLFR469 3.1 36.4 39.3 11.7 OLFR470 −0.8 7.1 2.7 7.1 OLFR478 −3.7 13.76.3 13.7 OLFR48 3.9 46.6 −8.7 11.9 OLFR486 0.7 35.5 −3.7 35.5 OLFR490−3.4 29.3 3.5 29.3 OLFR5 0.7 29.1 42.2 29.1 OLFR504 −5.3 11.2 56.6 11.2OLFR510 −0.9 9.4 39.5 9.4 OLFR516 3.4 25.8 31.5 7.6 OLFR517 0 24.8 0.824.8 OLFR524 0.9 9.7 35.6 9.7 OLFR525 −3.9 16.2 6.2 16.2 OLFR530 −3.316.4 −3.8 16.4 OLFR535 −1.5 20.5 9.7 20.5 OLFR536 −0.4 9 0.7 9.0 OLFR538−0.5 10.7 54 10.7 OLFR558 −0.9 8.5 −3.8 8.5 OLFR56 −11.9 48.4 −6 48.4OLFR566 −3.1 7.1 −2.5 7.1 OLFR57 −4.7 18.4 11.3 18.4 OLFR578 −2.8 8.825.8 8.8 OLFR597 0.7 9.2 74.5 9.2 OLFR606 2.8 26.5 −5.2 9.5 OLFR608 −4.613.5 18.5 13.5 OLFR616 −7.8 7.4 −3.6 7.4 OLFR618 6.6 54 78 8.2 OLFR619−2.3 8.2 24 8.2 OLFR62 −5.9 15.3 29.7 15.3 OLFR630 −0.2 13 −1.7 13.0OLFR638 −3.4 18.8 1.4 18.8 OLFR639 −1.4 12.2 11.7 12.2 OLFR64 −2.7 9.922.8 9.9 OLFR653 −1 8.4 15.7 8.4 OLFR654 0.9 21.2 −4.6 21.2 OLFR658 −112.1 14.4 12.1 OLFR659 3 42.3 69.1 14.1 OLFR665 2.3 48.3 83.9 21.0OLFR672 −3.9 12.5 −1.6 12.5 OLFR677 −0.5 15.5 89.1 15.5 OLFR681 3.4 27.30.9 8.0 OLFR691 −6.2 22 −2.7 22.0 OLFR692 1.4 18.9 64.2 13.5 OLFR698 0.613.6 4 13.6 OLFR702 3 50.9 83 17.0 OLFR710 0.2 11.3 5.1 11.3 OLFR716−0.3 13 −13.1 13.0 OLFR722 −0.1 8.3 23.6 8.3 OLFR725 −2.2 10.9 −0.9 10.9OLFR731 0.6 8 0.5 8.0 OLFR744 −3.5 7.5 31 7.5 OLFR748 −1.3 49.1 111.449.1 OLFR76 0.9 8 −2.4 8.0 OLFR761 1.8 14.3 −3.3 7.9 OLFR781 0.5 29.61.3 29.6 OLFR786 2.8 67 65.3 23.9 OLFR796 2.8 22.8 62 8.1 OLFR800 −3.311.2 28.6 11.2 OLFR812 −3.8 7.9 −3.4 7.9 OLFR816 −7.5 13.2 −3.7 13.2OLFR821 −5.9 9.1 5.4 9.1 OLFR824 −1.2 22.7 26.7 22.7 OLFR826 −2.6 7.511.5 7.5 OLFR828 −0.5 15.7 29.4 15.7 OLFR829 0.7 15.1 26.8 15.1 OLFR851−0.7 10.5 1.6 10.5 OLFR854 −4.2 17.5 −2.9 17.5 OLFR855 0.1 11.8 −2.611.8 OLFR868 −2.7 7.1 0.5 7.1 OLFR869 0 8.5 1.7 8.5 OLFR871 0.9 8 1 8.0OLFR872 −1.5 14 26.5 14.0 OLFR881 −7.3 7.8 10.2 7.8 OLFR906 −5 61.4 −7.261.4 OLFR910 4.7 86 69.5 18.3 OLFR912 −4.8 8.9 −3.6 8.9 OLFR917 2.5 25.50.1 10.2 OLFR923 −2.6 27.1 13.6 27.1 OLFR951 0 14.9 14.1 14.9 OLFR974−3.2 9.5 13.1 9.5 OLFR976 −4.4 12.3 3.9 12.3 OLFR980 −6.6 9.9 20.8 9.9OLFR983 −1.8 20.6 13.3 20.6 OLFR987 −1.2 9.2 −4.2 9.2 OLFR99 0.9 10.919.2 10.9 OLFR995 −1.6 9.1 3.5 9.1 vomeronasal 1 receptor V1RA2 −5 11.376.5 11.3 V1RB9 −5.6 8.6 5.8 8.6 V1RC10 −10.7 9.5 −5.1 9.5 V1RC8 −4.19.9 18.4 9.9 V1RD12 0 13.2 34.5 13.2 V1RD2 3.1 38.7 38.3 12.5 V1RD20−4.3 9.7 4.4 9.7 V1RD3 1.6 29 7.6 18.1 V1RE12 −3.1 33.6 −10.4 33.6V1RE13 −6.9 19.6 −6.1 19.6 V1RF3 −0.3 9.6 2.4 9.6 V1RG3 −1 11.1 34.711.1 V1RH13 −6 9.3 20.1 9.3 V1RH21 −3.3 19.8 51 19.8 V1RI1 −5.8 13.3−4.7 13.3 V1RJ3 −3.2 8.8 2.2 8.8 V1RL1 −3.9 8.1 35.2 8.1 Proteinsassociated with the cartilage CHST11 12.7 125.3 126.3 9.9 ASPN 0.2 141.8 14.0 ACAN 0.7 16 7.6 16.0 ADAM1B 1.3 14.6 −2.9 11.2 ADAM22 −2.7 19.27.3 19.2 ADAM29 −1.3 14 3.3 14.0 ADAM32 0.4 58.5 162.5 58.5 ADAM7 1 8.512.1 8.5 ADAMTS1 2 19.9 46 10.0 ADAMTS17 −3.2 21.7 −2.6 21.7 ADAMTS419.5 1742.5 2573.8 89.4 ADAMTS9 −1.2 11.4 30.1 11.4 ADAMTSL1 6 50 38.68.3 MMP12 −1.4 46.1 1.2 46.1 MMP13 6.9 284.6 539.4 41.2 MMP20 −2.4 13.4−2.5 13.4 MMP24 2.3 36.4 112.2 15.8 MMP24 −4.3 7.5 29 7.5 MMP27 −5.9 7.12.6 7.1 MMP3 3.7 54.5 3.9 14.7 MMP3 −0.6 9.9 16.1 9.9 TIMP1 326.5 3621.63232 11.1 TIMP1 383.6 3723.8 3477.1 9.7 TIMP3 11.1 87.9 142.7 7.9Protocadherin PCDH10 −0.9 89 90.9 89.0 PCDH10 −1.4 9.9 11.2 9.9 PCDHA111.4 42.8 14.2 30.6 PCDHA7 −1 10.6 −3.3 10.6 PCDHA7 6.1 50 98 8.2 PCDHB62 14.1 29.5 7.1 PCDHGA10 1.8 17.8 55.1 9.9 PCDHGA9 −0.5 11.8 0.7 11.8PCDHGB6 −4 7 −8.3 7.0 PCDHGB7 −2.9 8.3 62.3 8.3 PCDHGB8 −3.1 16.3 7.816.3 Prolactin family PRL2C2 −3.4 20.1 6.9 20.1 PRL2C3 −3.5 8.2 21.6 8.2PRL3D3 1 10.7 40.5 10.7 PRL4A1 −5.4 7.6 −2.5 7.6 PRL7A2 −6.8 7.6 −5.37.6 Prolactin receptor PRLR 22.5 173.6 257.9 7.7 TNF and TNF-relatedgenes TNF 1 854.3 94.3 854.3 0.5 21.4 4.2 21.4 TNF 3 1170.4 143.3 390.1−4.2 9.3 17.7 9.3 TNFAIP2 685 9076.5 1738.9 13.3 TNFAIP2 951.4 10471.82463.2 11.0 TNFAIP2 1876.9 15829.8 5120 8.4 TNFAIP3 42.3 2753.3 1202.965.1 0.5 21.4 4.2 21.7 TNFSF10 −4.6 40.8 111.2 40.8 TNFRSF10B 20 104.728.5 5.2 −4.2 9.3 17.7 38.9 TNFRSF10B 12.1 41.2 21.9 3.4 −4.1 21.7 −5.721.7 TNFRSF12A 307.7 1962.6 1503 6.4 TNFRSF1B 19.7 62.1 36.3 3.2 TNFSF114.5 66.1 35.1 14.7 TNFSF12-TNF 2.6 20.4 10.3 7.8 TNFSF13B −0.9 7.2 1.27.2 TNFSF14 −4.3 29.5 74 29.5 TNKS 3 84.9 116.2 28.3 TNFSF9 TNIP1 9.440.4 8.4 4.3 TNIP1 149.9 601.5 219.4 4.0 TRAF1 12.6 52.6 22.7 4.2 TRAF637.9 96.8 48.3 2.6 BAT5 10.4 89.8 31.3 8.6 Nuclear receptor subfamilyNR4A2 8.1 97.3 97.2 12.0 NR4A3 1 30.7 25.5 30.7 NR4A3 1 28.9 23.8 28.9NR5A1 1 6.5 4.6 6.5 circadian clock CCRN4L 7.7 141.7 141.6 18.4 GolgiGCC2 6.2 317 209 51.1 COG8 7.5 165.3 246.6 22.0 GOLGA2 30.5 599.1 1711.719.6 antioxidant CP 48.1 715.1 581 14.9 CP 22.9 253.6 233.9 11.1oncogenes VAV1 57.1 427.1 634 7.5 other tumor supressor H19 39.4 579.81096.1 14.7 immunomodulators LST1 97.1 721 772.9 7.4 chaperon ERAF 16148.7 190 9.3 Unclassified PRF1 1.4 74.9 23.9 53.5 ADAMTS1 42 139.8202.9 3.3 ADAMTS1 8.9 21.2 24.1 2.4 ADAMTS4 78 1034.4 945.4 13.3 ADM48.3 267.2 78.7 5.5 AI987692 1346.8 2785.5 3643.1 2.1 AIM1L 669.4 1706.51315.2 2.5 AKAP1 1 2.5 4.8 2.2 AKAP12 1202.9 2614.9 1811.3 2.2 AKAP214.3 53.9 48.5 3.8 AKAP2 100.2 247.1 193.1 2.5 AKAP2 399.6 915.1 718.72.3 AKAP4 1 5.6 0.7 5.6 AKAP8 1 2.3 3 2.3 AKR1B8 1874.5 5175.9 1788.22.8 AMD2 96.7 197 157.4 2.0 ANGPTL4 1 4.3 8.8 3.8 ANKRD1 7.2 35.5 63.24.9 ANKRD1 7.2 35.5 63.2 4.0 ANKRD11 1 6.8 3.4 6.8 ANKRD2 1 3.5 −3.3 3.5ANKRD56 104.9 314.3 104.2 3.0 ANKRD6 3.3 9.2 −0.3 2.8 ANKRD6 1 2.5 −0.12.5 ANKRD9 1 10.8 12.7 10.8 ARG1 1 16.7 28.7 16.7 ATP10D 374.9 773.6 4472.1 AXUD1 66.1 490.3 404.6 7.4 B230378H13R 5.9 34.2 24.7 5.8 BACH1 31.466.7 174.8 2.1 BCAR3 5 29.7 10 5.9 BDH1 1.9 10.6 0.3 5.6 C330006D17R 2.918.2 44.5 6.3 C330006P03R 207.3 618.3 403.5 3.0 C730046C01R 16.7 35.213.5 2.1 CCDC155 1 4.4 4 4.4 CCDC21 17.5 36.2 12.8 2.1 CCDC49 7.2 14.28.7 2.0 CCDC85B 2.1 130.3 52.5 62.0 CCDC89 1 4.7 5.1 4.7 CCDC93 4.2 10.52.7 2.5 CCDC94 84.3 203.1 121.4 2.4 CCDC99 31.4 71.4 50.2 2.3 CCT7 411.4 14.4 2.9 CDC5L 1 7.5 17.2 7.5 CDCA4 90.6 245.5 147.2 2.7 CDCA5 6.218.3 16.2 3.0 CLN5 1788.2 3680.5 2055.8 2.1 COQ10B 489.8 1789.3 1122.33.7 COQ10B 584.7 1758.4 1329.9 3.0 CTPS 322.7 978.8 860.3 3.0 CYR61 17.453.3 61.7 3.1 DGAT2 1832.3 6757.6 1508 3.7 DONSON 1.5 11.9 2.4 7.9DONSON 19.8 62.6 27.5 3.2 EDG7 64.1 403.6 103 6.3 EDG8 6.1 12 13.6 2.0ERRFI1 1168.7 4991.4 4278.4 4.3 F2RL1 208.8 1549.4 350.7 7.4 F2RL1 125.7850.8 158 6.8 FAR1 313.7 779.8 367.1 2.5 GCH1 110 1580 555.6 14.4 GM8264.8 75.6 6.3 15.8 GNL3 41.6 115.8 92.2 2.8 GRWD1 211.7 456.4 345.7 2.2GTLF3A 12.8 40.6 4.2 3.2 HDC 47.5 137.9 190 2.9 HP 56.6 508.4 240.5 9.0HP 39.2 334.8 190.8 8.5 HP 128.6 938.3 494.3 7.3 IDS 7.8 21.8 25.2 2.8IHH 1 23.2 −1.4 23.2 INS1 5.2 11.8 8.2 2.3 INSIG1 62.2 217.4 120.6 3.5INSIG1 83.1 221.6 159.4 2.7 INSR 1 8.7 3.6 8.7 IRG1 1 14.1 34.9 14.1IRG1 6.1 48.6 102.2 8.0 IRGQ 1 13.6 4.9 13.6 IRGQ 10.6 57.5 21.1 5.4IRX1 1 4.1 6.9 4.1 IRX2 8.6 16.3 7.5 7.5 IRX3 17.9 49.4 70.7 2.8 IRX4 12 −7.1 2.0 JMJD3 260.2 539 472.4 2.1 KLHL25 48.3 383.8 131.5 7.9 LDLR48.5 136.3 108.9 2.8 LIPK 7.4 50.6 13.5 6.8 MARCKSL1 76.6 162.6 131.82.1 MARCO 3.1 11.9 13.7 3.8 MAT2A 33.7 107.7 132.4 3.2 MAT2A 135.1 412.6463.5 3.1 MAT2A 60 173.3 214.4 2.9 MAT2A 2963.8 5883.3 4696.7 2.0 MATN42.5 6.8 6.6 2.7 MCM10 1 9.3 3.7 9.3 MCM10 5.9 42.9 25.5 7.3 MCOLN2 12.842.3 26.9 3.3 MFSD2 203.8 1933.3 339.4 9.5 MID1 14.1 48.5 9.7 3.4MOBKL1A 2.9 15.9 3.6 5.5 MOBKL2A 79 209.2 159.9 2.6 MOGAT2 22.2 72.520.5 3.3 MOGAT2 6.7 16 −3.8 2.4 MPPED1 3 21.8 12 7.3 MRGPRA2 5.5 20.20.4 3.7 MT1 11776.7 23326.9 25806 2.0 MT2 45.3 178.4 186.2 3.9 MTMR1459.2 173.6 111.9 2.9 MTMR14 50.8 118.4 56.5 2.3 MVD 423.3 1508.7 468.83.6 MYBBP1A 97.9 191.9 157.2 2.0 MYD116 256.8 1849 537.3 7.2 MYOM2 4.227.9 15.6 6.6 NFXL1 1.9 7.8 4.8 4.1 NFYA 14.6 44.3 19.2 3.0 NGFB 8.661.8 37.5 7.2 NGFB 19.7 86.7 61.4 4.4 NHLRC3 13.4 30.5 27.3 2.3 NLE1 115.7 18.3 15.7 NOC3L 136.1 295 175.7 2.2 NOL1 44.3 88.2 73.8 2.0 NRG139.5 105.4 92.9 2.7 NRIP3 8.8 30 20.5 3.4 NSUN5 27.7 58.5 42.9 2.1 NUAK2132.7 507.5 460.4 3.8 NUPR1 1049.6 3339.3 2496 3.2 OASL1 4.9 25.2 57.15.1 OASL1 4.4 16.9 41.2 3.8 PCDH10 1 38.4 7.4 38.4 PDE4A 6.2 20.2 13.13.3 PDE4B 4.6 23.1 13.7 5.0 PDE4B 1 4.9 2.1 4.9 PDE4B 30.9 145.3 168.44.7 PDE6G 2.3 4.7 5.7 2.0 PDGFB 2.2 15.1 4.6 6.9 PDGFB 120 249.1 168.82.1 PELI1 314.1 658 421.6 2.1 PELI3 1 11.7 −2.6 11.7 PHLDA1 1471.3 35471808.2 2.4 PIGM 1.5 12.6 11.5 8.4 PIGN 3.5 9.5 11.2 2.7 PIK3C2A 20.843.5 40.6 2.1 PLCXD3 1.1 7.3 11.7 6.6 PLEK 6.2 28.6 55.8 4.6 PLEKHG224.9 63.3 88.1 2.5 PLEKHG2 355 760.5 964.9 2.1 PLEKHG2 598.3 1260.31525.7 2.1 PMAIP1 19.1 60.4 32 3.2 PMAIP1 165.7 381.4 177.7 2.3 PMP22232.3 460.7 379.6 2.0 POF1B 460.9 953.8 732.6 2.1 PPAN 22.3 48.7 45.62.2 PRDM2 69.9 202.9 219 2.9 PRDM2 114.9 251.5 296.5 2.2 PRR7 34.5 767.7178.9 22.3 PRSS22 342.8 1971 767.7 5.7 PRSS23 1 6.5 8.3 6.5 PRSS27 27.7100.2 114.8 3.6 PTGES 44.1 239.1 96 5.4 PTGES 194.3 757.7 372.2 3.9PTGFRN 25.8 53.6 32.9 2.1 PTPN12 19.7 60.3 30.8 3.1 PTPN12 22.5 65.933.4 2.9 PTPN12 33.1 74.1 34.1 2.2 PTPN12 32.6 71.3 26 2.2 PTPN2 57.9217.3 86.2 3.8 RAMP3 1.1 14.1 17.1 12.8 RCAN1 34.6 371.1 242.1 10.7RDH10 17.9 64.4 21.9 3.6 RCAN1 34.6 371.1 242.1 10.7 RDH10 17.9 64.421.9 3.6 RETNLG 1 35.3 −4.3 35.3 RRP1B 4 12.4 6.7 2.5 RUNX1 53.9 116.763.8 2.2 SBNO2 70.3 319.7 167.2 4.5 SFN 1052.7 2514.4 1776.1 2.4 SGK11164.7 3136.8 2751.7 2.7 SH3BP2 196.1 504.2 219.7 2.6 SHROOM3 90.2 290.3161.2 3.2 SKIL 12.2 35.1 16.5 2.9 SNAI3 48.8 1451.2 204.6 29.7 SNF1LK17.1 150.4 61.8 8.8 SPSB1 660.2 2016.3 2569.9 3.1 ST3GAL1 23.1 101.333.9 4.4 STK35 17.8 60 25.6 3.4 SYNGR2 361.8 726.5 650.7 2.0 TAC1 1 17.819.5 17.8 TAL1 1 13.9 13.9 13.9 TAX1BP3 8.6 19.8 10.2 2.3 TBC1D10A 212.5553.3 285.6 2.6 TBC1D10A 437.9 948.2 531.3 2.2 TGIF1 31.2 134.8 25.2 4.3TGM1 1.4 17.6 28.1 12.6 THBS1 20.6 110.9 169.3 5.4 TIMP1 269 1445.71953.2 5.4 TREX1 2.6 8.7 9.2 3.3 TRIM21 6.2 18.5 16.6 3.0 TRIM27 27 79.350.7 2.9 TRPV4 58.1 183.4 73.2 3.2 TRPV4 78.2 244 109.8 3.1 TSSK6 5.738.2 8.8 6.7 TTC39B 14.7 40.4 36.9 2.7 UPP1 18.5 38 43.6 2.1 VPS37B915.9 2307.5 1154.6 2.5 WDR4 37.6 89 68.5 2.4 WDR43 371.1 803 494.6 2.2WDR46 3.9 8.2 −1.8 2.1 WDR70 8.1 16.3 12 2.0 WDR82 1 4.4 3.6 4.4 WDR9217.8 70.1 27.7 3.9 WNT10B 7.6 45.7 29.1 6.0 WNT7B 133 353.4 187.2 2.7WSB1 540.6 1289.7 909.7 2.4 YBX3 4821.9 17559.2 16672.8 3.6 ZFP295 33.980.4 47 2.4 ZFP36 901.1 6682.9 7114.2 7.4 ZFP57 13.2 68.7 21.8 5.2ZFP607 55 114.5 60.6 2.1 ZSWIM4 192.5 656.7 272 3.4 ARMC1 7.3 96.4 94.613.2 CHD7 15.3 172.1 251.4 11.2 CHKA 208.4 2673 3980.2 12.8 CRISPLD2334.1 2824.7 1873.8 8.5 EEF1A2 12.3 122.8 12.4 10.0 FABP5 41.1 435.8309.8 10.6 FBN1 13.9 106.9 47.4 7.7 FFAR2 27.2 208.4 4.9 7.7 FGL1 −2.474.7 260.5 74.7 FGL1 5.6 291.4 550.8 52.0 FIBP 5.5 76.2 203.5 13.9 GFPT114.6 105.7 33.8 7.2 GFPT2 97 1762.3 2584.6 18.2 GIMAP5 8.7 62.1 109.77.1 GMIP 69.8 489.2 536.8 7.0 HRC 19.8 148.3 238.2 7.5 ITIH4 9.2 86.989.5 9.4 MT2 53.7 1757.4 1431.7 32.7 MTRF1 7.6 72.6 −5.3 9.6 PCNP 233.22364.7 3003.8 10.1 PHF11 14.4 139.4 576.2 9.7 PHF11 12.9 98.8 431.3 7.7PIGR 10.4 153.5 102.4 14.8 RGS16 32 858.6 429.7 26.8 RHBDL2 95.2 1060.71284.9 11.1 SCIN 59.7 1853 145.9 31.0 SLA 57.8 430.4 435.4 7.4 SLAMF838.1 326.6 404.5 8.6 SNTB2 18.6 134.7 243.7 7.2 SP5 15.1 184.8 272 12.2SPINK8 14 123.3 161 8.8 SRR 253.8 3474.8 5827.9 13.7 SRR 150.9 1930.82623.3 12.8 TCOF1 78.8 675.2 1224.3 8.6 TIMELESS 40.9 411.4 684.6 10.1TIMM13 16 117.2 143.3 7.3 TSLP 4.1 64.1 118.5 15.6 TYKI 78.6 2634.13096.2 33.5 ATG4D 6.6 67.1 92.5 10.2 ATP5O 0.5 139.8 169.2 139.8 BACE228.8 262.8 229.6 9.1 BRIP1 9.5 109.1 101.8 11.5 CALCA −0.2 197.2 195.9197.2 CH25H 28.9 463.5 789.5 16.0 CRYBG3 12.6 94.6 159.7 7.5 DDAH1 11117 45.1 10.6 EPSTI1 7.7 111.5 39.6 14.5 GNG13 15 110.1 281.5 7.3LOC100047934 384.8 2880.7 2006.3 7.5 LOC100047963 69.1 989.1 857.8 14.3LOC100048556 26.2 705.6 1454.3 26.9 LOC638301 100 1318.6 1562.7 13.2NEK6 10.7 94.1 37.8 8.8 OSMR −0.8 62.9 −1.7 62.9 PCNP 233.2 2364.73003.8 10.1 SCL0002368.1_75 304.6 2626.6 3372.7 8.6 STFA1 9.9 93.1 559.4 STFA2 4.9 114.2 115.8 23.3 APOLD1 1 23.1 147.7 23.1 BC037703 1 36.7−2.7 36.7 CH25H 21 567 574.2 27.0 NOS2 8.8 101.4 11.2 11.5 0.1 108.8−7.6 108.8 ZC3H12A 3.8 590.8 84.5 155.5 123.5 2475.7 671.7 20.0 PLAT#REF! 5226.9 823.1 10.2 PLAUR 83.4 679.9 377.2 8.2 PSORS1C2 1 74.1 −8.774.1 −6.7 130.3 0 130.3 F10 2.2 7.2 63.5 3.3 DLL1 56.1 324.4 149 5.8DLL4 1 6.6 8.2 6.6 DLL4 1 3.9 1.1 3.9 Unknown function A630077B13R

1.1 58 39.4 52.7 A230065H16R

1 46.6 −6.3 46.6 1190003J15RI

1581.5 4188 2241.8 2.6 1500041J02RI

19 46.2 32.2 2.4 2310014H01R

2716.3 5339.4 3951.4 2.0 2310016C08R

319.3 2877.1 665.7 9.0 2310016C08R

319.3 2877.1 665.7 6.1 2210008F06RI

1 26.1 1 26.1 AI607873 73.4 188.4 182.8 2.6 AI987692 1346.8 2785.53643.1 2.1 AIM1L 27.2 76.5 33.5 2.5 2310014H01RIK 2310016C08RIK2310016C08RIK 2310026J01RIK 2310061F22RIK 2410025L10RIK 2810402K13RIK3830432E14RIK 5033413D16RIK 5530400B01RIK 5830457O10RIK 9930023K05RIK9930122J16RIK A130051J06RIK A130082M07RIK A230065H16RIK LOC10004623

266.4 882.3 962.3 3.3 LOC10004726

129.2 851 168.6 6.6 LOC10004733

42.2 87.6 88.8 2.1 LOC10004777

14.4 75.2 10.6 5.2 LOC10004793

613 2978.1 1464.3 4.9 LOC10004855

13.8 61.5 223.4 4.5 LOC10004855

27 91.5 373.9 3.4 LOC212399 254.5 965.7 432.3 3.8 LOC240672 86.8 713.8227.5 8.2 LOC381140 360.4 724.8 865.7 2.0 LOC638301 97.3 325.3 417.1 3.3D17H6S56E-3 4.3 15.9 10.5 3.7 D17H6S56E-5 30.4 580 90.8 19.1D330008I21RI

16 167.9 28.4 10.5 D7BWG0611E

1.3 27.1 8.8 20.8 D930038O18R

2.4 17.2 7 7.2 D930039D09R

1 11.1 3.7 11.1

indicates data missing or illegible when filed

1-31. (canceled)
 32. A method of treating a cancer present in a tissuethat expresses a Toll-Like Receptor (TLR), comprising administering aneffective amount of a TLR agonist to a subject in need thereof.
 33. Themethod of claim 32, wherein the TLR agonist is flagellin.
 34. The methodof claim 32, wherein the cancer does not express TLR.
 35. The method ofclaim 32, wherein the TLR is TLR5.
 36. The method of claim 32, whereinthe tissue is selected from liver, lung, bladder, or intestinal.
 37. Themethod of claim 32, wherein the cancer is metastatic.
 38. The method ofclaim 37, wherein the metastatic cancer is selected from melanoma,colon, breast, prostate, or a hematological malignancy.
 39. The methodof claim 38, wherein the hematological malignancy is lymphoma.
 40. Themethod of claim 32, wherein the cancer is a tumor.
 41. The method ofclaim 32, wherein the TLR agonist is administered as a monotherapy. 42.The method of claim 32, wherein the subject is not receiving acombination cancer therapy.
 43. The method of claim 32, wherein thesubject is not receiving chemotherapy or radiation therapy.
 44. Themethod of claim 32, wherein the subject has sufficient innate immunity.45. The method of claim 44, wherein the sufficient innate immunity levelis equivalent to the level required for eligibility for a first orsubsequent round of chemotherapy.
 46. The method of claim 32, whereinthe subject has a white blood cell count that is within the clinicallynormal range.
 47. The method of claim 32, wherein the TLR agonist isadministered to the subject before, after, or concurrent with removal ofa tumor.
 48. The method of claim 32, TLR agonist is co-administered witha FAS agonist.
 49. The method of claim 48, wherein the FAS agonist is aFAS agonist antibody.
 50. A method of reducing recurrence of a cancerpresent in a tissue that expresses a Toll-Like Receptor (TLR),comprising administering an effective amount of a TLR agonist to asubject in need thereof.
 51. The method of claim 50, wherein the cancerrecurrence is selected from a metastasis or a tumor regrowth.
 52. Themethod of claim 50, wherein the TLR agonist is flagellin.
 53. The methodof claim 50, wherein the cancer does not express TLR.
 54. The method ofclaim 50, wherein the TLR is TLR5.
 55. A method of treating a metastaticcancer that does not express Toll-Like Receptor (TLR) but is present ina tissue that expresses a TLR5, comprising administering an effectiveamount of a flagellin to a subject in need thereof.